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Granzyme B, a New Player in
Activation-Induced Cell Death, Is
Down-Regulated by Vasoactive Intestinal
Peptide in Th2 but Not Th1 Effectors
Vikas Sharma, Mario Delgado and Doina Ganea
J Immunol 2006; 176:97-110; ;
doi: 10.4049/jimmunol.176.1.97
http://www.jimmunol.org/content/176/1/97
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Copyright © 2006 by The American Association of
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References
The Journal of Immunology
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,† and Doina Ganea2*
ollowing 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
F
*Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
†
Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas, Granada, Spain
Received for publication April 28, 2005. Accepted for publication October 19, 2005.
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.).
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: [email protected]
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.
Copyright © 2005 by The American Association of Immunologists, Inc.
(Fas) (5, 6). Although both Th1 and Th2 effectors are ultimately
eliminated, Th1 cells are more susceptible to AICD (7–9). 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 –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 VIPinduced 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
0022-1767/05/$02.00
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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. The Journal
of Immunology, 2006, 176: 97–110.
98
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
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.
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
Macroarray
The expression of apoptotic genes in Th2 cells treated with or without VIP
was compared using the Panorama mouse apoptosis gene array (SigmaGenosys) 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
Reagents
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 ␮g/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 ⫻ 105 cells/
ml; total volume, 1 ml) with APCs (3 ⫻ 105 cells/ml) pulsed with 5 ␮M
PCCF. In the second experimental system, CD4⫹ T cells (1 ⫻ 106 cells/ml;
total volume, 2 ml) were seeded in 12-well tissue culture plates containing
immobilized anti-CD3 (3 ␮g/ml) and anti-CD28 (1 ␮g/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 ␮g/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 ␮g/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 ␮g/ml). The Th1 and Th2 effectors were restimulated with immobilized anti-CD3 Abs (10 ␮g/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 ␮g/ml) and APCs of polarized Th1 and Th2 effectors (5 ⫻
5
10 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).
Northern blot for FAP-1
Total RNA was isolated from Th1 and Th2 effectors (1 ⫻ 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 ⫻ 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, antiFasL, or goat anti-mouse GrB (2.5 ␮g/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 ␮g/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 ␮g/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
Cytokine ELISA
The contents of IL-4 and IFN-␥ 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-␥, R4-6A2/XMG1.2. The lower limits
of detection for IL-4 and IFN-␥ were 0.1 and 0.2 ng/ml, respectively.
CD4⫹ and CD8⫹ T cells were fixed and permeabilized using the Cytofix/
Perm kit, followed by incubation with rabbit anti-mouse GrB (2.5 ␮g/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 ␮g/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
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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 (21–27) was a gift from Drs. A. Welton and D. R. Bolin (both from
Hoffmann-LaRoche, Nutley, NJ).
Total RNA was extracted from 5 ⫻ 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⬘-CAAAGACTGG
CTTCATGTCCATT-3⬘, (reverse) 5⬘-GCAGAAGAGGTGTTCCATTGG
3⬘, and probe, 5⬘-FAM-ACAAGGACCAGCTCTGTCCTTGGCAG
TAMRA-3⬘. The primers and probe for ␤-actin are as follows: (forward)
5⬘-CGTGAAAAGATGACCCAGATCA-3⬘, (reverse) 5⬘-CACAGCCTG
GATGGCTACGT-3⬘, and probe, 5⬘-FAM-TTTGAGACCTTCAACAC
CCCAGCCA-TAMRA-3⬘. TaqMan RT-PCR was conducted with the onestep 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⬘-CGACGAGTTCGA
AGACCATTTT-3⬘; VPAC2, (forward) 5⬘-GGACAGCAACTCGCCT
CTCT-3⬘, (reverse) 5⬘-AGAATGGGCATCCGAATGAC-3⬘; EP-2, (forward) 5⬘-TGCAAGAGTCGTCAGTGGCT-3⬘, (reverse) 5⬘-AACAGTGC
CAGTGCGATGAG-3⬘; EP-4, (forward) 5⬘-TTTCTTCGGTCTGTC
GGGTC-3⬘, (reverse) 5⬘-CGCTTGTCCACGTAGTGGCT-3⬘; and Pfr,
(forward) 5⬘-CACGGCAGGGTGAAATTCTC-3⬘, (reverse) 5⬘-CCATGC
CAAGTGTCTGCCCC-3⬘. The primers for Bcl-2 are as follows: (forward)
5⬘-TGTGTGTGGAGAGCGTCAACA-3⬘ and (reverse) 5⬘-GATGCCG
GTTCAGGTACTCAGT-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.
The Journal of Immunology
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-CD3restimulated 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 ⌬␺ (membrane potential). Intact mitochondria accumulate more dye and emit higher fluorescence. To detect
mitochondrial integrity, the cells were incubated with 5 ␮l of TMRE dye
for 60 min and analyzed by flow cytometry.
A total of 2 ⫻ 106 PBS-washed viable cells was resuspended in 50 ␮l 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 ⫻ g). Five microliters of each lysate was used for
cell lysates (5 ␮l) 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 (AcIETD-pNA; BIOMOL), was used at 200 ␮M 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 ␮l. 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
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 PCCFspecific 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-␥ 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 al-
most 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).
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 (21–25). Both agonists
protected Th2 effectors from AICD (Fig. 2B), suggesting that both
VPAC1 and VPAC2 mediate the protective effect of VIP.
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 (EP2/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 VIPinduced 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
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GrB enzyme assay
99
100
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
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.
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.
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 antiCD3 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
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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). ⴱⴱ,
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.
The Journal of Immunology
101
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.
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.
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).
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).
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
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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
␤-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-MecAMP (50 –200 ␮M), followed by restimulation with immobilized antiCD3 Abs. Apoptosis was determined
by TUNEL 24 h later. The results are
the mean ⫾ SD of three independent
experiments. NT, Nontreated cells.
102
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
Table I. Changes in the expression of apoptosis-related genes in CD3restimulated Th2 effectors treated with VIPa
Expression Level
2
⫺1.5
2.5
2.3
1.7
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹
⫹
⫹⫹
2.6
3.6
2.1
2.9
3.2
3.7
2.1
2.5
2.4
3
2.4
2.5
3.3
2.2
2.9
2.7
4
2.1
5
2.1
2.2
2.2
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹⫹⫹⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
a
Th2 effectors were preincubated with or without VIP (10⫺7 M) for 30 min
followed by restimulation with immobilized anti-CD3 Abs for 3 h. [33P]cDNA was
used for array hybridization. The arrays were performed twice for each sample with
similar results. No changes were observed at 3 h in Bad, BAK, APAF-1, BID,
caspases, FasL, Fas, TRAIL, OX40, and CD40. ⫹⫹⫹⫹, Highly expressed; ⫹, low
expression.
(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 AcDEVD-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).
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 antiCD3 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.
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 ␤-actin message in
the same sample. One representative experiment of four is shown.
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-8induced 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-VADFMK 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
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Apoptotic gene
FLIPL/Cash (*)
Granzyme B (*)
cIAP-1
XIAP
Bcl-2
Apoptosis-related factor
ABP1
CAV2
CD47
CIDE-A
CLDN3
Cln3
Cox-2/Ptgs2
GPX1
GAPDH (*)
HD
ICAD/DFFA
MT-2
eNOS
nNOS
RP105/Ly78
SARP-2/sFRP-1
TFAR15
Thrombospondin
TIAF1
TIAL1
TSSC3
TXN
Fold Change
The Journal of Immunology
103
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
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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.
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 antiCD3 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
104
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
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).
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.
tible 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 antiCD3 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.
Both signaling through Fas and induction of GrB play a role in
AICD
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
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 suscep-
GrB produced by TCR-restimulated Th1 effectors kills activated
Fas-mutant CD4⫹ T cells
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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.
The Journal of Immunology
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 GrBInh 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).
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).
Although Fas/FasL-induced apoptosis is calcium independent,
EGTA could affect other apoptosis-related mechanisms. The de-
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.
livery 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
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
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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 ⫻ 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.
105
106
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
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
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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 ␮l; 1.5
␮g protein/ml) were preincubated for 30 min with the
pancaspase inhibitor Z-VAD-FMK (20 ␮M) or with the
GrB inhibitor I (20 ␮M), followed by the addition of
the GrB substrate (Ac-IEPD-pNA; 200 ␮M) or the
caspase-3 plus caspase-8 substrate (Ac-IETD-pNA and
Ac-DEVD-pNA, respectively; 200 ␮M) 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 ␮M), the neutralizing
anti-FasL Ab (10 ␮g/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. ⴱⴱ, Statistically significant (p ⬍
0.005). C, Th2 effectors were treated with etoposide (10
␮M) in presence of the pancaspase inhibitor Z-VADFMK (10 ␮M), GrB inhibitor I (20 ␮M), 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 ␮M), the GrB inhibitor I (20 ␮M), 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.
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 – 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 Journal of Immunology
107
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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.
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.
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 ␮g/ml) for 24 h. Lpr Th2
effectors were treated with the agonistic anti-Fas Jo.2 Ab (20 ␮g/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 ␮M) 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.
108
VIP DOWN-REGULATES GrB IN Th2 EFFECTORS
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
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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, antiCD3, 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 ⫻ 105 cells/ml) were cocultured
for 24 h in the presence or absence of
EGTA (5 mM) or GrB-inhibitor (20
␮M). 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 Journal of Immunology
109
Disclosures
The authors have no financial conflict of interest.
References
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
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as reference for apoptosis percentages. The results are the mean ⫾ SD of
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