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*
Laboratoire d’Immunodifferenciation, Université Denis-Diderot Paris 7, Paris, France; and
Institute for Immunology, University Hospital, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mono(ADP-ribosyl)transferases (ARTs)3 catalyze a posttranslational modification of proteins by transferring the ADP-ribose moiety of NAD to specific amino acids, e.g., arginine residues, on target proteins. These enzymes have well-known regulatory functions in the prokaryotic world and usually inactivate the function of target proteins (1). They also correspond to various bacterial toxins that have potent effects on mammalian cells (2). More recently, several ARTs have been cloned from different tissues. Five groups of ART-encoding genes, denoted ART1 to ART5, have been identified in the mouse, rat, rabbit, and human, based on sequence homology and genomic organization (3). ART1–ART4 isoforms correspond to 30- to 40-kDa GPI-anchored cell surface ARTs, whereas ART5 is not GPI-linked and may be secreted (4, 5). Their biological function is not fully understood, but they seem to play an important role in the regulation of myogenesis or long-term potentiation in hippocampal neurons, for instance, or in the regulation of lymphocyte functions (5, 6).
The first evidence supporting a role of ART in the immune system came from experiments in rats. In this species, there is a single locus encoding ART2 with two alleles (ART2a and ART2b), both expressed as 25- to 35-kDa GPI-anchored maturation markers on the surface of T lymphocytes (7). ART2+ T cells have been found to exert a regulatory role in models for autoimmune insulin-dependent diabetes mellitus. Indeed, diabetes prone BB-DP rats have reduced ART2 expression, and the transfer of ART2+ T cells from their BB-DR-resistant counterparts confers protection. Failure to develop this T cell subset is thus one of the genetically determined factors leading to enhanced susceptibility for autoimmune diabetes (8).
The mouse homologue of the rat ART2 gene has been tandemly duplicated to produce two loci (Art2a and Art2b) with multiple alleles (9, 10). In mice, ART activity has also been detected on lymphoid cells, including T cell lymphomas (11, 12), splenocytes (13, 14), and activated CTL (15) and is believed to participate in the regulation of cell functions. A 35-kDa GPI-anchored ART was found to mediate inhibition of proliferation and cytotoxic activity of CTL cell lines in the presence of extracellular NAD (16). A similar NAD-mediated inhibitory effect has more recently been reported on the proliferation of normal peripheral T cells in vitro (17). ARTs encoded by Art1, Art2a, Art2b, Art3, Art4, and Art5 can potentially mediate these effects, although neither Art5 nor Art3 are expressed on normal lymphoid cells (5, 18, 19). ART1 is a GPI-anchored protein that has been considered as a candidate ART responsible for this regulation because it was cloned from T cell lymphomas (20). However, ART1 is expressed at low levels in lymphoid tissues and predominates in heart and skeletal muscles (21). By contrast, Art2a and Art2b mRNA are expressed at higher level in lymphoid tissues (9, 10). Moreover, the recent development of specific mAbs has allowed us to demonstrate that ART2.2 is selectively present on mature T lymphocytes from most mouse strains although at different levels (22).
Deficient expression of ART2.1 and ART2.2 has been reported in several inbred mice. In C57BL/6, the Art2a sequence contains a stop codon leading to the synthesis of a truncated protein with no enzymatic activity (23). Reciprocally, NZW mice suffer from a deletion of the Art2b gene (24). Like in rats, defective expression of ART2s has been proposed as one of the factors influencing the onset and/or progression of autoimmune diseases (24, 25).
To date, most experiments concerning the role of ARTs and the effect of extracellular NAD on T lymphocytes have been performed in C57BL/6 mice that express ART2.2 at a very high level but lack functional ART2.1 on their surface (22, 23, 24). The potential role of differential ART expression on NAD-induced regulation of immune functions has not been investigated. Experiments reported herein address this issue by comparing the effect of extracellular NAD on T lymphocytes from appropriate inbred mice. We demonstrate that NAD triggers rapid induction of apoptosis in normal peripheral T cells from BALB/c and (C57BL/6 x NZW)F1 mice but not from natural knockout mice for ART2a (C57BL/6) or ART2b (NZW).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC-conjugated anti-CD3
(145-2C11) and PE-conjugated
anti-B220 (RA3-6B2) mAbs used for fluorescence staining were
purchased from BD PharMingen (San Diego, CA). FITC-conjugated
anti-CD8a (CT-CD8a) and PE-conjugated anti-CD4 (CT-CD4) were
obtained from Caltag Laboratories (South San Francisco, CA).
Animals
BALB/c and C57BL/6 mice were maintained in the animal facilities of the Institut Jacques Monod (Paris, France) and of the University Hospital (Hamburg, Germany). NZW/olaHsd mice were purchased from Harlan (Oxon, U.K.). F1 hybrids from C57BL/6 females mated with NZW males were bred and are designated elsewhere as B6NWF1. The offspring of the reciprocal mating were also tested and gave similar results (not shown). The Wistar rat R8 and the outbred rabbit 12760 were obtained from and maintained at the central animal facility of the University Hospital.
Antibodies
Preimmune sera were prepared from blood obtained by retroorbital bleeding from rat R8 and by draining of the ear vein from rabbit 12760 before ballistic DNA immunization with 8–12 shots (1 µg DNA coated on 1-µm gold particles) of pME.ART2.2, an expression vector for GPI-anchored, FLAG-tagged ART2.2, using a gene gun (Bio-Rad, Hercules, CA). Three booster immunizations were performed at 4- to 6-wk intervals. Animals received a final boost of purified recombinant ART2.2-human IgG1Fc fusion protein (50 µg in 500 µl PBS i.v.). Five days later, animals were sacrificed by heart puncture and immune sera were prepared. The specificity of antisera was determined by indirect immunofluorescence analyses using EL4 lymphoma cells and EL4 cells transfected with expression constructs for mouse ART1, ART2.1, or ART2.2. Immune sera 12760 and R8 specifically reacted with ART2.1 and ART2.2 transfectants but not with ART1 transfectants. ART2.2-specific rat IgG2a mAb (designated Nika-102) was generated following fusion of R8 splenocytes with Sp2/0 myeloma cells as described previously (22).
Cell preparation and proliferation assay
Splenocyte suspensions were prepared aseptically in cold PBS
containing 10% heat-inactivated FCS. Erythrocytes were removed by
treatment with NH4Cl (160 mM, 3 min). Cells were
then washed, resuspended in complete RPMI 1640 medium containing 5
x 10-5 2-ME, 0.2 mM glutamine, 1 mM pyruvate,
20 mM HEPES, and 10% FBS (all culture reagents were from Life
Technologies, Grand Island, NY). T cells were purified by two passages
over nylon wool columns. Purified T cells contained >80% living T
cells and <10% B cells as determined by flow cytometry. For in vitro
stimulation, spleen cells or purified T cells were cultured for 2 days
at 37°C in 96-well flat-bottom tissue plates (2 x
105 cells/well) either coated with 10 µg/ml
anti-CD3 (145-2C11) mAb or in the presence of 3 µg/ml Con A,
10 ng/ml PMA plus 100 ng/ml ionomycin (I0634; Sigma, St. Louis, MO), 4
µg/ml PHA, or 125 µg/ml LPS. Proliferative response was assayed by
adding 0.5 µCi/well of
[methyl-3H]thymidine (ICN
Pharmaceuticals, Costa Mesa, CA). After 8 h, cells were harvested
(Filtermate 196; Packard, Meriden, CT), and the radioactivity
incorporated into DNA was counted (TopCount; Packard).
Immunofluorescence analysis, detection of apoptotic cells, and treatment with phosphatidylinositol-specific phospholipase C (PI-PLC)
Standard procedures were used for immunofluorescence staining. Briefly, 1 x 106 cells were washed twice in FACS buffer (CellWash; Becton Dickinson, Mountain View, CA). They were then resuspended in 100 µl FACS buffer containing 1 µg of each mAb. After 30 min of incubation at 4°C, cells were washed twice and resuspended in 300 µl FACS buffer before analysis by flow cytometry on a FACSCalibur (Becton Dickinson).
Detection of apoptotic cells was performed by annexin V/propidium iodide (PI) staining as previously described (26). A second method was used to detect and quantify apoptosis based on TUNEL of DNA strand breaks (27). Briefly, cells were washed twice in PBS containing 1% BSA and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were washed at 4°C and permeabilized with sodium citrate buffer containing 0.1% Triton X-100 for 2 min on ice. After washing, cells were incubated with FITC-dUTP in the presence of TdT for 1 h at 37°C using the Cell Death Detection kit (Roche Diagnostic Systems, Somerville, NJ). Following incubation and washing, cells were counterstained with PI (0.05 µg/ml) in FACS buffer containing 200 µg/ml DNase-free RNase (Roche Diagnostic Systems). After 30 min of incubation at room temperature, cells were analyzed by flow cytometry.
In some experiments, lymphocytes were treated with PI-PLC before incubation with NAD and quantification of apoptosis using annexin V/PI staining. Cells were incubated for 30 min at 37°C in RPMI 1640 (2 x 107 cells/ml) containing 10 U/ml PI-PLC (Sigma). They were then suspended at 1 x 106 cells/ml in complete RPMI 1640 and incubated 1 h at 37°C with or without NAD before detection of apoptosis.
ADP-ribosylation of proteins
ADP-ribosylation of membrane proteins was analyzed by SDS-PAGE after incubation of cells with [32P]NAD as previously described (17) with some modifications. Briefly, 2 x 106 purified T lymphocytes were incubated in 100 µl complete RPMI 1640 medium containing 10 µCi [32P]NAD (350 µCi/mmol; ICN Pharmaceuticals), 1 mM ADP-ribose (Sigma), 100 µM cold NAD, and 1 µl protease inhibitor cocktail (P8340; Sigma) at 37°C for 1 h. T lymphocytes were then washed three times in cold RPMI 1640 to remove unbound radioactivity. Crude cell lysates were obtained by suspension of cell pellets in lysis buffer (PBS containing 1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF), followed by incubation on ice for 30 min. Insoluble material was pelleted by centrifugation (12,000 x g at 4°C for 15 min). Supernatants were collected. Proteins were separated by SDS-PAGE (10% gel), and dried gels were exposed to x-ray films at -70°C for 4 days using intensifying screens.
RT-PCR analysis of Art1, Art2a, and Art2b expression
The relative expression of the Art1,
Art2a, and Art2b genes was analyzed by
semiquantitative RT-PCR analysis. Total RNA was extracted from either
2 x 107 purified T cells from BALB/c,
C57BL/6, NZW, and B6NWF1 mice or BALB/c skeletal
muscle using the RNA Plus kit (Quantum-Appligene, Strasbourg, France).
cDNA was synthesized from 3 µg of total RNA in a volume of 33 µl
using the first-strand cDNA synthesis kit and
oligo(dT)18 as primers (Amersham Pharmacia
Biotech, Uppsala, Sweden) according to manufacturer’s recommendations.
The cDNA concentration of all samples was adjusted after pilot PCR for
-actin using forward 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and reverse
5'-TAAAACGCAGCTCAGTAACAGTCCG-3' primers. PCR were conducted in
20-µl reaction volumes containing 1 µl cDNA, 1x PCR buffer, 2.5 U
Taq polymerase, 1.5 mM MgCl2 (all from
Bioline, London, U.K.), 100 µM dNTPs (Roche Diagnostic Systems) and 5
pmol of each primer. PCR was performed on a PTC-200 thermocycler (MJ
Research, Cambridge, MA) for 30–40 cycles of 92°C, 60°C, and
72°C, 1 min each. Specific primers were derived from separate exons
in nonconserved regions of Art1, Art2a, and
Art2b deduced from the published sequences (GenBank
accession numbers X95825 (Art1); X52991 (Art2a),
and X87612 (Art2b)). Enzymatic digestion confirmed the
specificity of all PCR products (not shown). The following primers were
used: for Art1, forward ADPRT
5'-TGCTGGCCTACACCGCCAAC-3' and reverse 5'-TCAACATCGGGTAAGTTGCT-3'
or forward ADRT 5'-GAGCCACCGATCTCGCGGGTG-3' and reverse
5'-AGTTGACCAGCCTTCTTCAGTC-3'; for Art2a, forward
Art2a 5'-ATCCACAGAAGCCTTAATGAG-3' and reverse
5'-CTACGGCTCAGCAAGAGTAA-3'; for Art2b, forward
Art2b 5'-CCTCGCTATAGATTTTAACAG-3' and reverse
5'-CTACGGCTCAGCAAGAGTAA-3'. PCR products were analyzed on 1% agarose
gels and ethidium bromide staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before analyzing the effect of extracellular NAD on lymphocytes,
we determined the expression of Art2a-, Art2b-,
and Art1-encoded ART isoforms on nylon-purified T cells from
BALB/c, C57BL/6, NZW, and B6NWF1 mice by RT-PCR.
As illustrated in Fig. 1
, Art2a mRNA was present in T cells from BALB/c mice and, at
an apparently lower level, in cells from NZW and
B6NWF1 animals. In C57BL/6, a low
Art2a signal was detected, but this sequence contains an
in-frame stop codon as reported previously (23).
Art2b was transcribed at high level in C57BL/6 mice but at
lower level in BALB/c and in B6NWF1 hybrids. This
was consistent with results obtained by staining with ART2.2-specific
Nika-102 mAb as already reported (22). As expected,
Art2b transcripts were absent in ART2.2-deficient NZW mice
(24).
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Ecto-NAD selectively inhibits T cell activation
It has previously been reported that extracellular NAD suppresses
the proliferation of C57BL/6 mouse T cells. We wondered whether the
differential expression of Art2a and Art2b would
influence the sensitivity to NAD-mediated suppressive effects on T cell
functions. To address this question, we first compared the effects of
extracellular NAD on spleen cell proliferation induced by Con A in
BALB/c and C57BL/6 mice (Fig. 2A). In both strains, cell
proliferation was strongly inhibited by low concentrations of NAD with
an IC50 in the range of 3–5 µM. The
proliferative response was also blocked by NAD in
B6NWF1 mice, whereas NZW T cells were resistant.
The effect of NAD on BALB/c splenocytes was specific to T cells, as
previously reported for C57BL/6 lymphocytes (17), because
LPS-induced B cell proliferation was not affected (Fig. 2B) We further tested the effect of NAD on the proliferation
of nylon-purified T cells induced by Con A, PHA, anti-CD3 mAb, or
PMA-ionomycin. Fig. 2C shows that NAD inhibited the response
irrespective of the activation pathway, including the direct
stimulation of cytoplasmic protein kinase C by PMA and ionomycin. This
indicated that the effect of NAD, at least in BALB/c mice, was not
caused by an early blockage of the transmembrane signaling pathway.
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D). NZW
T lymphocytes were much less affected even after 48 h. An
intermediate and slower toxicity of ecto-NAD was observed on C57BL/6 T
cells. At 48 h, cell viability was rather similar in all mice
except NZW. This was in good agreement with the inhibitory effect of
ecto-NAD on T cell proliferation, but suggested a differential
mechanism of toxicity between C57BL/6 and BALB/c or
B6NWF1 mice.
To test whether CD4 and CD8 subsets were equally sensitive to NAD
toxicity, BALB/c spleen cells were incubated with 50 µM NAD for
48 h and were then analyzed by flow cytometry (Fig. 3). In the absence of NAD, the CD4:CD8
ratio was close to 2. With 50 µM NAD, most T cells were eliminated
but the CD4:CD8 ratio remained unchanged. This demonstrated that
extracellular NAD has a direct toxic effect on both naive
CD4+ and CD8+ T lymphocytes
in the absence of additional stimulus. The results also confirmed that
B220+ B cells were not sensitive to NAD
toxicity.
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The rapid toxic effect of NAD on naive T lymphocytes from BALB/c
and B6NWF1 mice could result from necrosis or
from the induction of programmed cell death. To address this issue,
purified splenic T cells from BALB/c or C57BL/6 mice were incubated for
1.5 h with NAD and then stained with annexin V/PI. As illustrated
in Fig. 4A, NAD at 10 µM
induced apoptosis of T lymphocytes from BALB/c but not from C57BL/6
mice. T cell apoptosis was dependent on the NAD concentration added to
cultures (Fig. 4B) and could be detected as early as 10 min
after incubation (data not shown). C57BL/6 mice could thus be
considered as resistant to NAD-induced apoptosis as previously
suggested (17). Analysis of DNA strand breaks by TUNEL
after 6 h of incubation confirmed that BALB/c T cells were
sensitive to NAD-induced apoptosis (Fig. 4C). Again, only a
small fraction of C57BL/6 T cells were engaged into programmed cell
death. Taken together, these observations indicated that ecto-NAD
blocks T cell activation in both BALB/c and C57BL/6 mice, but
selectively induces rapid apoptosis in BALB/c mice. As expected from
their resistance to NAD-mediated inhibition of T cell proliferation,
NZW T cells were also resistant to NAD-mediated apoptosis.
Interestingly, apoptosis was induced in B6NWF1 T
cells, with similar kinetics as in BALB/c, although both their C57BL/6
and NZW parents were resistant (Fig. 4). This demonstrated that at
least two genetic factors are involved in NAD-induced apoptosis.
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The observation that NAD-induced apoptosis in BALB/c and in
B6NWF1 T cells raised the question of the
molecular mechanism underlying this effect. Extracellular NAD cannot
penetrate into cells but can be degraded by several enzymes, including
NAD-glycohydrolases such as CD38 (29) and
phosphodiesterases such as PC-1 (30). These enzymes can
transform NAD into metabolites that could deliver signals of programmed
cell death (31). These metabolites include adenosine, ADP,
AMP, nicotinamide, ADP-ribose (ADPR), and cyclic ADP-ribose (cADPR). To
test the possibility that the toxic effect of NAD was mediated by one
of these metabolites rather than by NAD itself, purified T lymphocytes
from BALB/c mice were incubated for 30 min with NAD metabolites, and
the relative number of apoptotic cells was evaluated. As illustrated in
Fig. 5A, apoptosis was induced
by incubation with NAD but not by any other NAD metabolites or
derivatives tested. This demonstrated that the apoptotic signal was
dependent on a surface protein acting as a direct NAD acceptor. The
role of P1 purine receptors, which bind adenosine, and of P2 purine
receptors, which bind ATP, ADP, UTP, and to a lesser extent NADP
(32, 33), was ruled out. Results also excluded the
possible intervention of CD38, which is only expressed on a small
fraction of murine T lymphocytes (34), because no effect
was observed with ADPR or cADPR. As also shown in Fig. 6, double staining of
B6NWF1 T cells with CD38 and annexin V after
incubation with increasing concentrations of NAD revealed that T cells
engaged into apoptosis belonged to the CD38-
population and that CD38+ cells were
resistant.
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B
support this prediction by showing that removal of GPI-anchored
proteins leads to concomitant loss of sensitivity to extracellular
NAD. Direct involvement of ART activity in the apoptotic process
We took advantage of a recently developed ART2.2-specific mAb and
of ART2-specific antisera to evaluate the functional role of ART2 and
ADP-ribosylation in NAD-mediated T cell apoptosis. In a first series of
experiments, we used Nika-102, a mAb that binds ART2.2
(22) but does not inhibit its enzymatic activity (data not
shown), to follow cells undergoing apoptosis in the presence of NAD.
Nylon-purified T cells were incubated with different concentrations of
NAD for 1.5 h and then double stained with Nika-102 and annexin V.
As illustrated in Fig. 7, 
66% of T
cells from B6NWF1 mice were
ART2.2+. With increasing concentrations of NAD,
the number of cells undergoing programmed cell death increased
concomitantly with a diminution of the ART2.2-expressing population.
Furthermore, annexin V+ cells were largely
restricted to the Nika-102+ population. By
contrast, the fraction and number of ART2.2-
cells, which were not labeled by annexin V, remained unchanged. Similar
results were obtained with BALB/c T cells (not shown). These
observations demonstrated that the cells committed to apoptosis by NAD
were only those expressing ART2.2 on their surface.
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). As illustrated below
(Fig. 9), this was consistent with the
ability of these Abs to inhibit ART2-transferase activity. Together,
this demonstrated that NAD-mediated apoptosis requires ART2-mediated
ADP-ribosylation of membrane proteins.
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The presence of ART(s) on naive T cells predicts that incubation
with radioactive NAD should result in labeling of cell surface
proteins. To test this prediction, nylon-purified T lymphocytes
isolated either from natural knockout mice for Art2a
(C57BL/6) or Art2b (NZW) or from mice expressing
Art2a and Art2b (BALB/c and
B6NWF1) were incubated with
[32P]NAD for 1 h. Crude cell lysates were
then prepared, and labeled proteins were analyzed by autoradiography
after SDS-PAGE. As illustrated in Fig. 9A (lane
2), strong ADP-ribosylation of several proteins was routinely
observed with cells from C57BL/6 mice, consistent with their high level
of ART2.2 expression (22). The pattern of proteins labeled
was very similar to that already reported by Okamoto et al. in these
mice (17). A similar pattern of bands was observed also in
cells from BALB/c and B6NWF1 animals (Fig. 9A, lanes 1 and 4), although the
intensity of labeling was lower than in T cells from C57BL/6 mice. In
marked contrast, little if any ADP-ribosylated cell surface proteins
were detected on cells from ART2.2-deficient NZW mice (Fig. 9A, lane 3). Preincubation of T cells with
ART2-specific antisera K12760 (Fig. 9, B and C)
or R8 (data not shown) reduced labeling of cell surface proteins to
background levels in all strains tested. These findings confirmed that
most if not all of the ART activity on resting murine T lymphocytes can
be attributed to the ART2 ADP ribosyltransferase.
Taken together, these results further suggest that resistance of NZW T cells to NAD-mediated apoptosis can be attributed to the virtual absence of cell surface ADP-ribosyltransferase activity on these cells. Similarly, T cells with strong cell surface ART activity, e.g., those of BALB/c and B6NWF1 mice, can be rendered resistant to NAD-mediated apoptosis by inhibiting ART activity with ART2-specific Abs. In contrast, resistance of C57BL/6 T cells to NAD-mediated apoptosis cannot be attributed to lack of ADP-ribosyltransferase activity. Instead, it seems most likely that these cells carry a defect in a downstream effector of ART activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This is consistent with previous studies
reporting an inhibitory effect of NAD on the proliferation of CTL lines
and of naive peripheral T cells (15, 16, 17). Yet, more
importantly, we demonstrate here that extracellular NAD has a direct
toxic effect on T cells from BALB/c mice and induces programmed cell
death in the absence of any additional cosignal (Fig. 4). This
contrasts with the situation in ART2.1-deficient C57BL/6 mice, in which
NAD blocks T cell activation but does not induce apoptosis
(17), and in ART2.2-deficient NZW mice, which are
refractory to both NAD-mediated apoptosis and inhibition of T cell
proliferation.
It has previously been shown that the suppressive effects of ecto-NAD
on T cells of C57BL/6 mice depend on GPI-anchored ART(s)
(15). Similarly, we demonstrate that removal of
GPI-anchored cell surface proteins by treatment of BALB/c T cells with
bacterial PI-PLC strongly reduces their susceptibility to NAD-induced
apoptosis (Fig. 5B). Implication of ART(s) rather than other
NAD-metabolizing ectoenzymes such as CD38 or PC-1 (29, 30)
is also attested by the observation that none of the NAD metabolites
tested can mediate apoptosis (Fig. 5A), that cells
undergoing apoptosis are ART2.2+ (Fig. 7), and
that apoptosis can be inhibited by polyclonal Abs to ART2.2 (Fig. 8).
In mice, at least six distinct GPI-anchored ARTs, namely ART1, ART2.1,
ART2.2, ART3, ART4, and ART5, have been identified that could be
involved in the apoptotic process (3, 4, 18, 19). ART1 has
been cloned by different groups from mouse lymphoma cells (Yac-1), from
a BALB/c skeletal muscle cDNA library, or from mouse genomic DNA.
Northern blot and RT-PCR analyses previously reported indicate that
Art1 is prominently expressed in cardiac and skeletal
muscles and at much lower level in lymphoid tissues (12, 20, 21). Although ART1 has been detected in T cell lymphomas and
hybridomas, direct evidence was still lacking for its expression on
normal peripheral T lymphocytes. Our attempt to detect ART1 by RT-PCR
on naive splenic T cells was negative even by using different pairs of
specific primers and irrespective of the mouse strain tested (Fig. 1).
It can thus be concluded that Art1 is not expressed on
normal T lymphocytes. Weak signals detected by others for
Art1 expression in lymphoid organs may be due to its
expression on other cell types (35). Alternatively,
Art1 expression may be developmentally regulated and limited
to fully differentiated T cells like activated CTL or to transformed T
cell lymphomas (20). A rather similar situation has been
reported for ART5, which was isolated from Yac-1 lymphoma cells but,
again, is not expressed on normal lymphoid spleen cells
(18). In contrast, our results confirm previous
observations (9, 22, 24) that Art2a and
Art2b transcripts are found in resting BALB/c T cells (Fig. 1). In C57BL/6 mice, a low level of Art2a transcripts is
detected, but this mRNA contains a stop codon that prevents the
expression of a functional enzyme (23). Moreover, our
finding that ART2-specific antisera abolish labeling of cell surface
proteins following incubation with [32P]NAD
(Fig. 9) supports the conclusion that the ART2s represent the
predominant ARTs expressed on normal peripheral T cells. It further
implies that ART2.2, and presumably also ART2.1, can ADP-ribosylate a
number of different cell surface proteins.
The very high sensitivity of T cells from B6NWF1
animals to NAD-induced apoptosis, whereas T cells of either parental
animal are resistant (Fig. 4), is important because it indicates that
complementation of (at least) two genetic factors is operative in cells
of the F1 animals in NAD-mediated apoptosis. We
propose that one of these is ART2 cell surface ADP-ribosyltransferase
activity. This hypothesis is supported by our findings that
ART2-specific antisera block both ADP-ribosylation of cell surface
proteins (Fig. 9) and NAD-mediated apoptosis of
B6NWF1 cells (Fig. 8). It is further supported by
our finding that cells from ART2.1/ART2.2 double-knockout mice are
completely resistant to NAD-mediated apoptosis (our unpublished
observations). The resistance of NZW T cells to NAD-induced apoptosis
(Fig. 4), then, could be attributed to the virtual absence of ART
activity on these cells (Fig. 9). Consistent with this interpretation,
we find that treatment of NZW T cells with DTT, which is known to
stimulate the activity of ART2.1 (13), enhances both
NAD-mediated ADP-ribosylation of cell surface proteins and apoptosis
(our unpublished observations).
We propose that the second genetic factor governing sensitivity to
rapid NAD-mediated apoptosis of resting T cells in
B6NWF1 animals concerns a downstream effector of
ADP-ribosylation. The observation that C57BL/6 T cells are resistant to
NAD-mediated apoptosis (Fig. 4) despite a high level of cell surface
ART activity (Fig. 9) implies that ART activity alone is not sufficient
for mediating apoptosis. We hypothesize that C57BL/6 mice are resistant
to NAD-mediated apoptosis due to a defect in a downstream effector of
ADP-ribosylation and that this defect is complemented in
B6NWF1 animals by the corresponding factor from
the NZW parent. Further analyses are required to determine whether this
factor is itself a target protein for ADP-ribosylation or a signaling
component activated by ART activity. In this context, it may be
interesting to note that lysates of BALB/c and
B6NWF1 T cells contain a prominent 35-kDa protein
that is labeled weakly in lysates of C57BL/6 mice (arrows in Fig. 9A).
The molecular mechanisms underlying rapid NAD-mediated apoptosis of
naive T lymphocytes are still unknown but several hypotheses can be put
forward. Activation of the caspase pathway is a well-established
mechanism for inducing apoptosis of T cells. However, preliminary
evidence using caspase inhibitors indicate that NAD does not activate
this pathway in resting T cells. Signaling through GPI-anchored
proteins has been reported in diverse settings (36). One
could thus postulate that ART2.2 itself, as a GPI-anchored protein,
would deliver the apoptotic signal to naive T cells. This possibility
is supported by the observation that RT6 is associated with tyrosine
kinases and could thereby, potentially, generate intracellular
signaling (37). Alternatively, a membrane protein distinct
from ART might be involved in apoptotic signal delivery. For example,
the apoptotic signal could be delivered via ADP-ribosylation of
important costimulatory signaling cell surface molecules. Indeed, CD45,
CD44, CD43, or CD27 have been shown to be ADP-ribosylated on
NAD-treated T cells (17). Conceivably, this could result
in inappropriate weak stimulation signals that in turn would cause
programmed cell death. It is indeed established that costimulatory
molecules are associated with protein kinases involved in signal
transduction (38). Such weak stimulatory signals have been
shown to induce apoptosis at different stages during lymphocyte
differentiation (39). However, our results indicate that
strong stimulation of cells with mitogenic activators fails to rescue
cells from death (Fig. 2). Recently, an additional mechanism involving
CD38 has been proposed to mediate apoptosis of activated T cells
(40). Indeed, ADP-ribosylation of CD38, by blocking its
cyclase activity and cADPR production, was shown to induce apoptosis in
T cells recently activated by soluble CD3 mAb (40).
However, our results indicate that resting T cells lacking CD38 are
sensitive to NAD-induced apoptosis whereas CD38-expressing cells are
resistant (Fig. 6). Conceivably, the apoptotic mechanisms induced by
NAD may differ in activated and nonactivated T cells.
Next to the remarkable rapid induction of apoptosis of resting T cells
described here, NAD evidently influences T cell functions in other
ways. This is supported by the finding that C57BL/6 mice are resistant
to rapid induction of apoptosis (Fig. 4), but are sensitive to
NAD-mediated inhibition of proliferation (Fig. 2). Given that several
different proteins are ADP-ribosylated following treatment of cells
with ecto-NAD (Fig. 9), it is not surprising that ecto-NAD affects T
cell functions in more than one way. As shown by Dennert and coworkers,
ADP-ribosylation of various membrane proteins affect T cell signaling
and functions via ADP-ribosylation of important cell surface molecules
including CD8 coreceptors and LFA-1 (16, 17, 41). This
inhibitory effect of NAD is consistent with the demonstration that
transfection of EL4 T lymphoma cells with Art1 results in
failure of TCR and coreceptors to associate into a functional
macromolecular complex in the presence of ecto-NAD as a consequence of
ADP-ribosylation (28).
Under physiological conditions, the concentration of NAD in extracellular body fluids is low, but it can increase in injured tissues due to the liberation of their intracellular content by dead cells. On the basis of our findings, it is tempting to speculate that NAD would then kill naive T cells present in these tissues or block their recruitment by nonprofessional APC. In contrast, recently activated, i.e., Ag-specific T cells, that have shed ART2 (14) would be resistant to the apoptotic effects of ecto-NAD. NAD-mediated apoptosis could thereby play a role in restricting the proliferation of bystander lymphocytes. This provides a plausible scheme for NAD to participate in the control of autoimmunity as already suggested by ART deficiency in mice and rats prone to autoimmune diseases (8, 24, 25).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michel Seman, Laboratoire d’Immunodifferenciation, EA 1556, Université Denis-Diderot Paris 7, CP 7124, Tour 54, 2 place Jussieu, 75251 Paris Cedex 05, France. E-mail address: seman{at}paris7.jussieu.fr
3 Abbreviations used in this paper: ART, mono(ADP-ribosyl)transferase; PI-PLC, phosphatidylinositol-specific phospholipase C; ADPR, adenosine diphosphoribose; cADPR, cyclic adenosine diphosphoribose; PI, propidium iodide.
Received for publication February 5, 2001. Accepted for publication April 27, 2001.
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