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NAD
ⴙ
Released during Inflammation Participates in T Cell
Homeostasis by Inducing ART2-Mediated Death of Naive T
Cells In Vivo
1
Sahil Adriouch,*
†‡
Sandra Hubert,*
†
Severine Pechberty,* Friedrich Koch-Nolte,
†‡
Friedrich Haag,
†‡
and Michel Seman
2
*
†
Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of
ADP-ribose groups from NAD
ⴙ
onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP
receptor and is responsible for NAD
ⴙ
-induced T cell death (NICD). Yet, the origin of extracellular NAD
ⴙ
and the role of
NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release
of NAD
ⴙ
into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining
lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD
ⴙ
glycohydrolase, whereas no effect is
observed in ART2-deficient animals. Intravenous injection of NAD
ⴙ
used to exacerbate NICD in vivo results in fast and
dramatic ART2- and P2X7-dependent depletion of CD4
ⴙ
and CD8
ⴙ
T lymphocytes, which can affect up to 80% of peripheral
T cells in CD38
ⴚ/ⴚ
mice. This affects mainly naive T cells as most cells surviving in vivo NAD
ⴙ
treatment exhibit the
phenotype of recently activated/memory cells. Consistently, treatment with NAD
ⴙ
abolishes primary Ab response to a
T-dependent Ag in NICD-susceptible CD38
ⴚ/ⴚ
mice but has no effect on the secondary response when given several days
after priming. Unexpectedly NAD
ⴙ
treatment improves the response in their wild-type BALB/c counterparts. We propose
that NAD
ⴙ
released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of
resting cells, thus contributing to the dynamic regulation of T cell homeostasis. The Journal of Immunology, 2007, 179:
186 –194.
Mammalian mono-ADP-ribosyltransferases (ART)
3
constitute a family of ectoenzymes structurally related
to bacterial toxins catalyzing the transfer of the ADP-
ribose group from NAD
⫹
onto amino acid residues of target pro-
teins (1–3). ART1–ART4 paralogs are GPI-anchored in the outer
leaflet of the plasma membrane and are thus responsible for unique
posttranslational modifications of proteins in the extracellular
compartment. Like phosphorylation mediated by protein kinases,
ART-mediated modification of proteins by ADP-ribosylation
leads to structural changes that can either inhibit or stimulate
target protein functions. ART activities in vitro can be split into
those relevant to trans-ADP-ribosylation of free soluble targets
such as cytokines and growth factors or proteins produced by
neighboring cells and those relevant to cis-ADP-ribosylation of
proteins present on the surface of the ART-expressing cells (3).
Although ADP-ribosylation of proteins can easily be shown in
vitro (2, 4 – 6), its reality and role in vivo are still poorly doc-
umented (7, 8).
In the mouse, two of the six ART paralogs, namely, ART2.1
and ART2.2, are expressed on the surface of most mature pe-
ripheral T lymphocytes (1, 9). It was first shown that a GPI-
anchored ART is expressed on the surface of mouse cytotoxic
T cell lines and that incubation of cells with NAD
⫹
leads to the
inhibition of both T cell proliferation and cytotoxic activity (6).
This inhibition was associated with ADP-ribosylation of several
membrane proteins such as LFA-1, CD8, CD27, CD43, CD44,
or CD45 (10 –12). Recently, the association of ART2 with lipid
rafts has been shown to focus ART2 on specific targets (13). It
was postulated that ADP-ribosylation of coreceptors inhibits
TCR-signaling by altering receptor association as well as cell
contacts and T cell trafficking (10, 12). We subsequently
showed that incubation with NAD
⫹
leads to the rapid induction
of ART-dependent T cell death in vitro detectable at NAD con-
centrations as low as 1
M, a phenomenon that we proposed to
call NAD
⫹
-induced T cell death (NICD) for NAD
⫹
-induced
cell death (4, 14). We also discovered that NICD results from
the activation of the P2X7 ATP receptor, which is another tar-
get of murine ART2 (14).
P2X7 belongs to the P2X family of ATP-gated ion channels
expressed on different cell types in the immune system including
T lymphocytes (15, 16). P2X7 activation triggers calcium flux,
shedding of CD62L, phosphatidylserine (PS) exposure, opening of
a large nonselective membrane pore and ultimately cell death by
apoptosis (17–19). Incubation of mouse T lymphocytes with
*University Denis Diderot-Paris 7, EA1556, Paris, France;
†
Institut National de la
Sante´ et de la Recherche Me´dicale Unite´ 519, Faculty of Medicine and Pharmacy,
Rouen, France;
‡
Institute of Immunology, University Medical Center, Hamburg-Ep-
pendorf, Hamburg, Germany
Received for publication December 20, 2006. Accepted for publication April
14, 2007.
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 grants from the Ligue Nationale Contre le Cancer, the
Association pour la Recherche sur le Cancer, and the Ministe`re de la Recherche (to
M.S.); the Fondation pour la Recherche Me´dicale (to S.A.), and the Deutsche
Forschungsgemeinschaft (to F.K.-N. and F.H.).
2
Address correspondence and reprint requests to Prof. Michel Seman, Institut Na-
tional de la Sante´ et de la Recherche Me´dicale, U519, Rouen, F-76000, France. E-mail
address: michel.seman@univ-rouen.fr
3
Abbreviations used in this paper: ART, ADP-ribosyltransferase; PI, propidium
iodide; NADase, NAD
⫹
glycohydrolase; ADH, alcohol dehydrogenase; PES,
phenazyne ethosulfate; PS, phosphatidylserine; NICD, NAD
⫹
-induced T cell death.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
micromolar NAD
⫹
concentrations induces all these effects,
whereas P2X7 activation by ATP requires millimolar concentra-
tions (14, 17). NICD depends on ART2 and P2X7 as assessed by
the resistance observed with T cells from ART2-deficient or P2X7-
deficient mice (14, 20, 21). Moreover, C57BL/6 T cells, which
harbor a P451L natural mutation in the C-terminal intracytoplas-
mic domain of P2X7 that severely impairs the response to ATP
(22), are relatively resistant also to NICD in vitro, further support-
ing the conclusion that P2X7 is required for NAD
⫹
-induced T cell
apoptosis.
The origin of the ecto-NAD
⫹
ART-substrate is still poorly
understood. The intracellular NAD
⫹
concentration is in the
range of 1 mM, whereas the plasma concentration is in the
range of 0.1
M, below the Km of ARTs (5, 23, 24). It is
speculated that high amounts of ecto-NAD
⫹
can be released
during tissue injury as a consequence of cell lysis. However,
nucleotides such as ATP or NAD
⫹
also seem to be released by
nonlytic processes under various physiological conditions in-
cluding hypoxia, inflammation, and mechanical or chemical ac-
tivation (25–27). Connexin 43 hemichannels, for instance, me-
diate transmembrane NAD
⫹
fluxes in intact cells (25). Hence,
high local NAD
⫹
concentrations may be reached under various
physiological and pathophysiological situations which would
permit ADP-ribosylation of membrane proteins on ART-ex-
pressing neighboring cells. The fate of this NAD
⫹
is controlled
by ecto-NADases. As recently shown, the ecto-NADase CD38
expressed on B lymphocytes, competes with ARTs for the
NAD
⫹
substrate, controls its availability and therefore the level
of ART2-catalyzed ADP-ribosylation on the surface of mouse T
lymphocytes (28). This regulation is likely to occur in vivo in
secondary lymphoid tissues where T and B lymphocytes are in
close contact.
The aim of the present study was to evaluate NAD
⫹
release
during inflammation and to explore its consequence on T cell pop-
ulations in vivo. We used a model of inflammation induced by
polyacrylamide beads to investigate NAD
⫹
release in exudates
during the course of an acute inflammatory response. We then
explored the effect of NAD
⫹
on the different T cell subsets in
lymph nodes drained by the inflammatory site or after systemic
injection of NAD
⫹
. Our results show that high amounts of NAD
⫹
are released during acute inflammation. They also demonstrate that
NAD
⫹
can induce strong T cell depletion in vivo and that cells
surviving NAD
⫹
treatment are enriched in activated/memory T
lymphocytes. We finally show that NAD
⫹
treatment in vivo affects
Ab production to T dependent Ags providing evidence that NICD
participates in T cell homeostasis in the course of the immune
response.
Materials and Methods
Mice
BALB/c mice were obtained from the Centre d’Elevage Janvier (Le Genest
Saint Isle, France). ART2-deficient mice were generated by standard ho-
mologous recombination procedures and backcrossed for 12 generations
onto the BALB/c background as described elsewhere (20). CD38-deficient
mice were gifts from Dr. F. Lund (Trudeau Institute, New York, NY) (29).
All mice were maintained under standard conditions in the animal quarter
of the Institute Jacques Monod (University Paris 7, Paris, France). Exper-
iments were performed according to European regulations.
Reagents and Abs
ATP, NAD
⫹
, propidium iodide (PI), Con A, and Neurospora crassa
NAD
⫹
glycohydrolase (NADase) were obtained from Sigma-Aldrich. PE-
and FITC-conjugated anti-B220, anti-CD3, anti-CD4, anti-CD8, anti-
CD25, anti-CD44, anti-CD69, anti-CD62L, anti-CD11b, anti-Gr1, and An-
nexin V
FITC
were from BD Pharmingen. ART2.2-specific Nika 102 mAb
(9) was purified from hybridoma supernatant by affinity chromatography
on protein G-Sepharose (Pharmacia) and conjugated to FITC (Sigma-
Aldrich). P2X7-specific Hano44 mAb was obtained by genetic immuniza-
tion of rats and fusion of spleen cells with Sp2/0 myeloma cells as de-
scribed previously (9, 30). Abs were purified from hybridoma supernatants
and conjugated to Alexa Fluor 488 (Molecular Probes) according to the
manufacturer’s instructions. SRBC were purchased from Eurobio.
Assay for phosphatidylserine exposure and cell death
Single-cell suspensions from lymph nodes were prepared and processed by
flow cytometry on a FACSCalibur instrument using the CellQuest-Pro pro-
gram (BD Biosciences) as described elsewhere (4, 9). B cells were depleted
by magnetic cell separation using anti-B220 Abs (BD Pharmingen) con-
jugated to Adembeads prepared according to the manufacturer’s instruc-
tions (Ademtech). Purity of T cells was always ⬎95% as verified by FACS
analysis using PE-conjugated anti-B220 and FITC-conjugated anti-CD3.
Following treatment with NAD
⫹
or ATP for 30 min at 37°C, cells were
washed in RPMI 1640 medium (Invitrogen Life Technologies) and were
resuspended in annexin V binding buffer (BD Pharmingen). They were stained
for 20 min on ice with FITC-conjugated annexin V (1
g/ml) and propidium
iodide (10
g/ml).
Inflammation
Inflammation was induced as previously described (31). Briefly, animals
were shaved and 800
l of a sterile 67% suspension (53 mg dry weight/ml)
of Biogel P100 (Bio-Rad) in PBS was injected s.c. into the bottom of the
back. At given times, pouches were incised on sacrificed animals. The
Biogel mass was collected under a dissecting microscope with a spatula
and resuspended in 0.5 ml of PBS/20 U/ml heparin. Tubes were weighed
before and after addition of the beads to determine the Biogel mass recov-
ered. After vigorous shaking to extract cells and exudate components from
the gel, beads were allowed to sediment and supernatants were collected.
Cells were then recovered by centrifugation and cell-free exudates were
immediately used for NAD
⫹
measurement or frozen and kept at ⫺20°C.
Total cells in exudates were counted on a hemocytometer. Cell subpopu-
lations were differentially evaluated by flow cytometry after staining with
PE-conjugated anti-Gr-1 and FITC-conjugated anti-CD11b.
NAD measurements
NAD
⫹
concentrations were determined by a cyclic enzymatic assay as
described previously (23). The first step of the cycle is an enzymatic re-
action catalyzed by the NAD
⫹
-dependent alcohol dehydrogenase (ADH).
The next steps are nonenzymatic reactions. NADH
2
produced during the
enzymatic step is oxidized by phenazyne ethosulfate (PES) which, once
reduced, is itself oxidized by thiazolyl blue (MTT). Oxidized NAD
⫹
and
PES are used in the next cycle. Absorbance of the reduced form of MTT
is measured at 570 nm in a two-chamber spectrophotometer. A “premix”
solution containing 3.2 ml of sodium-1.5 M (pH 7.8) bicine (N,N-bis(2-
hydroxyethyl)glycine) buffer, 2.4 ml of 10 M ethanol, 2.0 ml of 10 mM
MTT, 1.0 ml of 0.2 M EDTA, 0.4 ml of 100 mg/ml BSA, and 1.0 ml of
H
2
O was prepared. Measurements were performed by mixing in the dark
0.3 ml of premix solution with 0.1 ml of 40 mM PES, 0.1 ml of 1 mg/ml
ADH in sodium-bicine buffer (0.1 M; pH 8.8), and 0.1 ml of a standard
NAD
⫹
solution or sample. OD was followed over a period of 3–5 min.
NAD
⫹
concentration was deduced from standard curves. Determination of
the initial speed of the cyclic reaction, for each known concentration of
NAD
⫹
allowed us draw a standard curve: V⫽f[C], which then permits
accurate calculation of NAD
⫹
concentration in samples. We routinely used
initiation by NAD
⫹
for inflammatory exudates (colorless in our model) but
initiation by ADH for plasma. All reagents were purchased from
Sigma-Aldrich.
Immunization and Ab titration
Mice were immunized i.p. with 0.2 ml of a 10% sheep erythrocyte sus-
pension (Eurobio) in PBS (32). Serum samples were collected at appro-
priate times and hemagglutination titers were determined by serial dilutions
of 50
l of serum in PBS with 25
l of a 0.4% SRBC suspension.
Data representation and statistical analysis
Results are expressed as the mean ⫾SD. Significance was assessed by
Student’s ttest with pat least inferior to 0.05 indicating statistical
significance.
187The Journal of Immunology
Results
NAD
⫹
is released during acute inflammation
To evaluate whether NAD
⫹
is released during inflammation and to
determine its concentration in inflammatory exudates, we took ad-
vantage of the experimental model of acute inflammation induced
by s.c. injection of Biogel polyacrylamide beads (31, 33). In this
model, surgical recovery of the beads and their suspension in a
defined volume of PBS gives access to the number of cells and to
the real concentration of NAD
⫹
trapped per unit of gel mass re-
covered. This method, which is not traumatic for cells, allows the
determination of free NAD
⫹
concentration in inflammatory exu-
dates. Wild-type BALB/c mice were injected with Biogel and
sacrificed at various time points to follow the kinetics of NAD
⫹
concentration and cellular infiltration during the course of the in-
flammatory reaction (Fig. 1B). Cells recovered in the gel were
counted and NAD
⫹
concentration was determined by an enzy-
matic assay. In this model, maximal cell infiltration is achieved
after 24 h and up to 48 h and primarily correspond to neutrophils
as assessed by Gr-1/CD11b staining (Fig. 1A), which are then
progressively replaced by macrophages (31). Remarkably, free
NAD
⫹
was detected in exudates. The concentration peaked at 12 h
after induction of inflammation, a time when the cellular response
was still building up (Fig. 1B). This suggests that NAD
⫹
is ac-
tively released from cells as an early response to inflammatory
stimuli as opposed to being passively set free as a result of cell
lysis or neutrophil apoptosis.
The influence of CD38 and ART2 in this process was explored
by performing similar experiments in CD38 and ART2-deficient
BALB/c mice. Twenty-four hours after treatment with Biogel, the
number of neutrophils was lower in exudates from CD38
⫺/⫺
mice
compared with wild type (Fig. 2A). This is consistent with the
established role of CD38 in neutrophil chemotaxis and homing in
response to inflammatory stimuli (34). At peak, NAD
⫹
concentra-
tion reached ⬎10
M in exudates from CD38
⫺/⫺
BALB/c mice
but ⬍4
M in their wild-type or ART2
⫺/⫺
counterparts (Fig. 2B).
This indicated that the CD38 NADase participates in the clearance
of NAD
⫹
at the inflammatory site. Yet, NAD
⫹
release presented
the same kinetics as in wild-type animals with a peak at 12 h
preceding massive neutrophil influx in the pouch (Fig. 2C). NAD
⫹
release in exudates had no effect on the basal plasma level, which
remained unchanged throughout the inflammatory reaction (Fig.
2Band data not shown). Higher NAD
⫹
concentrations were found
in plasma of untreated (data not shown) and Biogel-treated
CD38
⫺/⫺
mice compared with wild-type animals. This may either
reflect elevated steady-state NAD
⫹
levels in these animals or have
resulted from NAD
⫹
released by hemolysis occurring during
blood collection and preparation.
Inflammation induces NICD in vivo
In vitro, micromolar concentrations of NAD
⫹
are sufficient to in-
duce rapid apoptosis of mouse T cells which can be detected by
annexin V/PI staining (4, 14). We, therefore, next tested whether
NAD
⫹
present in inflammatory exudates could induce cell death in
vitro. As shown in Fig. 3A, exudates collected after 12 h from
CD38
⫺/⫺
BALB/c mice induced significant apoptosis of purified
lymph node T cells. The effect was limited due to the dilution of
exudates that was necessary to extract the NAD
⫹
from the Biogel,
leading to a working concentration in the range of 1.5
M. The
notion that NAD
⫹
in inflammatory exudates caused PS flashing is
supported by the finding that PS flashing was inhibited by pretreat-
ment of exudates with exogenous NADase. Moreover, inflammatory
exudates did not induce any detectable annexin V/PI staining of
ART2-deficient T cells (data not shown).
These results prompted us to evaluate the effect of Biogel-in-
duced inflammation on T cells in lymph nodes from wild-type,
CD38
⫺/⫺
, and ART2
⫺/⫺
BALB/c mice. One day after injection of
Biogel beads s.c., in the bottom of the back, draining (inguinal and
para-aortic) and nondraining (axillary) nodes were separately col-
lected. Cells were counted and analyzed by flow cytometry. A
significant diminution of ⬎20% in the number of CD3
⫹
T cells
was observed in the draining nodes of wild-type-treated mice but
not in the nondraining axillary nodes of the same animals (Fig.
3B). A more severe reduction of ⬎50% was observed in the drain-
ing nodes from CD38
⫺/⫺
mice but no effect of inflammation was
observed in ART2
⫺/⫺
mice treated under the same conditions
(Fig. 3B). Together, these results demonstrated that NAD
⫹
re-
leased during inflammation can reach the draining nodes and can
FIGURE 1. NAD
⫹
release during acute inflammation. Inflammatory
pouches were induced in BALB/c mice with 0.8 ml of a 50% Biogel bead
suspension s.c. Cells and exudates were collected at the times indicated. A,
Phenotype of inflammatory cells collected after 24 h and stained with
CD11b/Gr-1 Abs. B, Kinetics of cell infiltration (E) and NAD
⫹
release
(⽧) in inflammatory exudates (n⫽6 mice/point). Error bars, SD.
FIGURE 2. Comparison of inflammatory response and NAD
⫹
release in wild-type, CD38
⫺/⫺
, and ART2
⫺/⫺
BALB/c mice. Inflammation was induced
as described in Fig. 1. A, Total cell number in inflammatory exudates 24 h after treatment with Biogel were determined in wild-type (WT; n⫽11), CD38
⫺/⫺
(n⫽11), and in ART2
⫺/⫺
mice (n⫽7). B, NAD
⫹
concentration in plasma and exudates at 12 h (n⫽6 for each group). C, Kinetics of cell infiltration
and NAD
⫹
release in exudates from CD38
⫺/⫺
mice (n⫽6). Error bars, SD.
188 NICD IN VIVO
induce T cell depletion in vivo. Consistent with previous reports
(28), CD38 evidently partially protects T cells from the ART2-
dependent deleterious effect of NAD
⫹
.
In CD38
⫺/⫺
mice, T cell depletion in the draining nodes was
accompanied by a relative increase in the fraction of CD62L
low
T cells (Fig. 4). This could reflect the resistance of CD62L
low
T
cells to NAD
⫹
-induced death and/or shedding of CD62L by
surviving cells as a consequence of P2X7 activation (14). When
10 mg of NAD
⫹
was mixed with Biogel, a more pronounced T
cell depletion and CD62L shedding was observed in draining
nodes but also in nondraining ones (Fig. 4). This confirmed that
NAD
⫹
released at local inflamed sites can reach draining lymph
nodes (see Fig. 4, Biogel, middle panels) and even nondraining
lymph nodes through lymphatic and blood circulations provided
that the NAD
⫹
amount is high enough (see Fig. 4, Biogel ⫹
NAD, right panels).
NAD
⫹
injection induces rapid T cell depletion in vivo
To further explore the effect of NAD
⫹
on T cell populations in
vivo, BALB/c mice were injected i.v. with the maximal tolerated
dose of either ATP (6 mg) or NAD
⫹
(10 mg) in 0.2 ml of PBS.
After 24 h, spleen and lymph nodes were recovered and the size of
the T cell population was evaluated by CD3/B220 staining in flow
cytometry. A significant reduction in the percentage of CD3
⫹
cells
was observed in lymphoid organs from NAD
⫹
-treated mice,
whereas ATP treatment did not have any significant effects (Fig.
5A). The differential effects of ATP and NAD
⫹
were consistent
with the higher concentration of ATP compared with NAD
⫹
re-
quired to induce T cell death by P2X7 activation in vitro (14) and
with the short half-life of ATP in body fluids due to widely dis-
tributed ecto-ATPases (35, 36). Reduction of the percentage of
CD3
⫹
cells was accompanied by a 40% drop in the absolute T cell
count in secondary lymphoid tissues (Fig. 5B). As expected, T cell
depletion was more dramatic in CD38
⫺/⫺
mice compared with
wild-type BALB/c mice, particularly in lymph nodes (Fig. 5C),
whereas NAD
⫹
treatment had no effect on T cells in their
ART2
⫺/⫺
counterparts (Fig. 5D). Evidently, NAD
⫹
-induced T
cell depletion in vivo is ART2 dependent, in accord with previous
observations in vitro (14, 20). Results in CD38
⫺/⫺
mice are in
agreement with our previous report demonstrating the antagonistic
effect of the CD38 NADase on ART2-mediated T cell death (28).
In CD38
⫺/⫺
BALB/c mice, T cell depletion was clearly detectable
as early as 3 h after NAD
⫹
injection and reached its maximum at
⬎70% after 1 day (Fig. 5E). NAD
⫹
treatment had little if any
effects on B cell counts in spleen and lymph nodes. The effect on
T lymphocytes was long-lasting and mice had not fully recovered
normal T cell numbers, 1 wk after NAD
⫹
treatment. These obser-
vations strongly suggested that NAD
⫹
induces rapid T cell death
in vivo as already described in vitro (4).
Cells resistant to NICD exhibit the phenotype of
activated/memory T cells
The effect of treatment with NAD
⫹
in vivo on different T cell
subsets was followed by examining cell phenotypes in lymph
nodes 24 h after the injection of NAD
⫹
into CD38
⫺/⫺
BALB/c
mice. The results indicate, first, that CD4
⫹
cells appear to be more
sensitive to NICD than their CD8
⫹
counterparts (Fig. 6,Aand B).
FIGURE 3. NAD
⫹
released in exudates induces T cell apoptosis in
vitro and T cell depletion in vivo. A, Induction of apoptosis in BALB/c T
cells by 12-h inflammatory exudates from CD38
⫺/⫺
BALB/c mice. Puri-
fied T cells were incubated with exudates or NAD
⫹
solutions in PBS for 30
min at 37°C and then stained with Annexin V
FITC
/PI. Right panel,5Uof
NADase was added to T cells just before addition of the exudate. No gate
was applied from the scatters plots to analyze living and shrinking/dying
cells. The numbers indicated in the lower right quadrant represent the
percentage of apoptotic cells defined as annexin V
⫹
/PI
⫺
. Results are rep-
resentative of six individual mice from three independent experiments. B,
T cell number in draining (para-aortic and inguinal) and nondraining (ax-
illary) lymph nodes in control or Biogel-treated wild-type (wt; n⫽12),
CD38
⫺/⫺
(n⫽12), and ART2
⫺/⫺
(n⫽6) BALB/c mice. Lymph nodes
were collected 24 h after induction of inflammation and cells were counted.
T cell numbers were deduced from the percentage of CD3
⫹
cells estimated
after CD3/B220 staining in flow cytometry. Vertical bars, SD and pvalues
are from Student’s ttest.
FIGURE 4. Analysis of T cells in draining and nondraining lymph nodes in CD38
⫺/⫺
BALB/c mice 24 h after induction of inflammation. Inflammation
was induced with 0.8 ml of a 50% Biogel bead suspension s.c. or Biogel containing 10 mg of NAD
⫹
. T cells from draining (para-aortic and inguinal) and
nondraining (axillary) nodes from CD38
⫺/⫺
BALB/c mice were collected after 24 h and stained with anti-CD3-FITC/anti-B220-PE or anti-CD3-FITC/
anti-CD62L-PE mAbs. Panels are representative of six mice from two independent experiments. Right panels, cells were gated on the CD3
⫹
population.
189The Journal of Immunology
Indeed, 78 ⫾11% of CD4
⫹
cells and 52 ⫾21% of CD8
⫹
cells
were affected in the lymph nodes of these animals 24 h after the
injection of NAD
⫹
(p⫽0.003, n⫽10). This could be accounted
for by a difference in ART2 and/or P2X7 density on the plasma
membrane. In accord with a previous report, we show that CD8
⫹
T cells express a higher level of ART2.2 on their surface than
CD4
⫹
cells as assessed by staining with ART2.2-specific Nika102
mAb (Ref. 9) and Fig. 6C). Yet, CD8
⫹
T cells display a lower
P2X7 receptor density than CD4
⫹
cells as assessed by P2X7-spe-
cific Hano44 mAb (Fig. 6C). This suggests that susceptibility to
NICD is more influenced by the density of P2X7 than of ART2 on
the cell surface.
Second, cells surviving NAD
⫹
treatment in vivo show striking
phenotypic differences to cells of sham-treated mice. Indeed, in-
creased percentages of CD44
high
and CD69
high
along with a de-
creased percentage of CD62L
high
T lymphocytes were observed
among cells resistant to NICD in vivo (Fig. 6D). These markers
characterize the pool of recently activated/memory T cells. Anal-
ysis of cell numbers in vivo reveals that NAD
⫹
treatment has no
direct effect on the size of the CD44
high
T cell population and only
a slight effect on the size of the CD69
high
population (Fig. 6E). Yet,
both populations seem to expand 10 days after treatment, a time at
which the peripheral T cell pool is being replenished. Our results
therefore indicate that NAD
⫹
treatment has a strong effect on na-
ive T cells and that activated/memory T lymphocytes are resistant
to NICD in vivo.
Analysis of ART2.2 expression after NAD
⫹
treatment, using
Nika102 mAb, reveals that a significant fraction of the CD4
⫹
cells
and most of the CD8
⫹
cells resistant to NAD
⫹
in vivo do not
express ART2.2 on their surface (Fig. 7A). Since activated T cells
shed ART2 from their surface (37), this along with their CD44,
CD62L, and CD69 phenotypes supports the conclusion that endo-
genously activated or memory cells are less sensitive to the dele-
terious effect of NAD
⫹
. Moreover, CD4
⫹
and CD8
⫹
cells resistant
FIGURE 5. Injection of free NAD
⫹
induces T cell
depletion in vivo. A, BALB/c mice were injected with
0.2 ml of PBS or 0.2 ml of PBS containing 6 mg of ATP
or 10 mg of NAD
⫹
. CD3/B220 staining was performed
on spleen and lymph nodes (13 nodes collected) 24 h
after injection. Profiles are representative of at least 12
mice. B, T cell numbers in spleen and lymph nodes from
BALB/c mice 24 h after treatment with NAD
⫹
. The
mean value indicated by the horizontal bars correspond
to 60 ⫾9⫻10
6
and 41 ⫾10 ⫻10
6
cells in the spleen
of PBS- vs NAD-treated mice (n⫽9, respectively, and
to 24 ⫾5⫻10
6
and 15 ⫾4⫻10
6
cells in the lymph
nodes of PBS- vs NAD-treated mice (n⫽12), respec-
tively. Statistical analyses were performed using Stu-
dent’s ttest. C, CD3/B220 staining in CD38
⫺/⫺
BALB/c 24 h after injection of 10 mg of NAD
⫹
. Results
are representative of 12 individual mice. D, CD3/B220
staining in ART2
⫺/⫺
BALB/c 24 h after injection of 10
of mg NAD
⫹
. Results are representative of five indi-
vidual mice. E, CD38
⫺/⫺
BALB/c mice were treated
with 10 mg of NAD
⫹
i.v., and total cell counts were
determined in spleen and lymph nodes at various times.
T and B cell numbers were deduced from the percent-
ages measured after CD3/B220 staining. Each point rep-
resents the mean of six individual mice.
190 NICD IN VIVO
to NICD in vivo are also poorly stained with Hano44 mAb com-
pared with cells from untreated mice (Fig. 7A), consistent with the
notion that sensitivity to NICD is greater for cells expressing high
levels of P2X7. Correlatively, we show that activation of murine T
cells by Con A (Fig. 7B) or by anti-CD3 Abs (data not shown)
leads to a down modulation of both P2X7 and ART2.2 on the
plasma membrane.
Extracellular NAD
⫹
influences the immune response to SRBC
The influence of NAD
⫹
on the immune response was tested in
wild-type, ART2
⫺/⫺
, and CD38
⫺/⫺
BALB/c mice after immuni-
zation with SRBC. This Ag was selected for several reasons. First,
SRBC Ab response is T dependent and quickly elicits detectable
primary and secondary serum Ab titers. Second, response to SRBC
does not require the use of adjuvants, which are inflammatory and
delay Ag delivery. In a first series of experiments, mice were
treated with 10 mg of NAD
⫹
i.v. at the time of immunization with
2⫻10
8
SRBC in saline i.p. Mice were bled after 6 days and
primary agglutination titers were determined. A much lower Ab
titer was observed in CD38
⫺/⫺
mice treated with NAD
⫹
vs un-
treated mice, whereas NAD
⫹
treatment did not show any detect-
able effect on Ab induction in ART2
⫺/⫺
mice (Fig. 8A). This is
consistent with the dramatic T cell depletion induced by NAD
⫹
in
CD38
⫺/⫺
animals and the resistance of T cells from ART2
⫺/⫺
mice to NICD in vivo. Conceivably, elimination of most naive T
cells by NAD
⫹
in CD38
⫺/⫺
mice affects the recruitment of Ag-
specific cells.
A second group of mice was treated with NAD
⫹
on day 4 after
priming, a time when the response was still building up. They were
then boosted with SRBC on day 14 and the secondary response
was tested on day 20. Agglutination titers were compared with
those in mice treated with NAD
⫹
on day 0 and to untreated mice.
Results in Fig. 8Bshow that treatment with NAD
⫹
on day 4 after
priming did not block the secondary SRBC response in CD38
⫺/⫺
mice or even slightly increased it in comparison to untreated
CD38
⫺/⫺
mice. Notice that the secondary response in mice treated
with NAD
⫹
on day 0 was equivalent to the primary one in un-
treated animals. These results further support the notion that NICD
affects naive T cells, whereas activated T cells escape the delete-
rious effect of NAD
⫹
.
Remarkably, different results where obtained in wild-type
BALB/c mice which are less sensitive to NICD than their
CD38
⫺/⫺
counterparts. Indeed, treatment with NAD
⫹
in these
mice led to a significant improvement of both primary and sec-
ondary responses compared with controls (Fig. 8). This suggests
that NAD
⫹
-induced partial T cell depletion of naive T cells, which
likely corresponds to physiological situations in vivo, may facili-
tate the expansion of Ag-primed T cells. Note that NAD
⫹
treat-
ment at day 14, i.e., immediately before the secondary antigenic
challenge, had no effect on the secondary response in wild-type or
in CD38
⫺/⫺
BALB/c mice (Fig. 8B). This result is consistent with
the idea that recently primed T cells responsible for the secondary
response are refractory to NICD.
FIGURE 6. T cell phenotype in CD38
⫺/⫺
BALB/c mice after treatment
with NAD
⫹
. CD38
⫺/⫺
BALB/c mice were treated with 10 mg of NAD
⫹
i.v. or 0.2 ml of PBS. A, Six peripheral lymph nodes were collected at
different times and total cells were counted. Cell numbers were calculated
after CD3-FITC/B220-PE and CD8-PE/CD4-FITC staining. Each point is
the mean of three individual mice. B, CD4:CD8 ratio among B220-de-
pleted lymph node cells 24 h after treatment with NAD
⫹
(representative of
seven mice). C, Comparison of P2X7 and ART2.2 expression on CD4
⫹
-
and CD8-purified T cells in untreated mice after staining with P2X7-spe-
cific mAb Hano44 or ART2.2-specific mAb Nika102. The mean fluores-
cence intensity (MFI) values were 33 vs 50 for P2X7 expression on CD8
⫹
vs CD4
⫹
cells, respectively, and 48 vs 21 for ART2.2 expression on CD8
⫹
vs CD4
⫹
cells, respectively. The MFI values of the isotype controls were
in the range of 5–7. These results are representative of at least four mice.
D, Phenotypes of CD4
⫹
lymph node T cells were determined before or
24 h after NAD
⫹
injection. Cells were gated on the CD4
⫹
population after
staining with CD4-allophycocyanin/CD62L-FITC or CD4-allophycocya-
nin/CD44-CyChrome/CD69-PE. Panels are representative of six individual
mice from three independent experiments. E, Cell numbers were calculated
as in Afrom the percentages observed after CD4/CD44/CD69 staining.
Each point is the mean of three individual mice.
FIGURE 7. ART2 and P2X7 expression after NAD
⫹
treatment in vivo.
A, Lymph node-purified T cells from control or NAD
⫹
-treated CD38
⫺/⫺
BALB/c mice were stained with PE-conjugated anti-CD4 or anti-CD8 and
counterstained with FITC-conjugated mAb Nika102 mAb for ART2.2 ex-
pression and Alexa Fluor 488-conjugated mAb Hano44 for P2X7 expres-
sion 24 h after treatment with NAD
⫹
or PBS. The MFI values in PBS vs
NAD treatment were, respectively, 50 vs 35 (P2X7 expression on CD4
⫹
);
33 vs 18 (P2X7 expression on CD8
⫹
); 28 vs 23 (ART2.2 expression on
CD4
⫹
); and 46 vs 23 (ART2.2 expression on CD8
⫹
). The MFI values of
the isotype controls were in the range of 5–7. Panels are representative of
three individual mice. B, Lymph node-purified T cells from wild-type
BALB/c mice were cultured for 24 h with Con A (2
g/ml) in RPMI
1640/10% FCS and stained for expression of P2X7 and ART2.
191The Journal of Immunology
Discussion
The discovery of mono-ART in vertebrates has opened a new field
in cell communication by introducing new ectoenzymes catalyzing
posttranslational modifications of proteins using NAD
⫹
in the ex-
tracellular compartment (3). ART1 and ART2 are presently the
two best-characterized paralogs (1), but their role in vivo remains
elusive. In mice, ART2 is expressed on most peripheral T cells and
remarkably induces P2X7 activation by ADP-ribosylation leading
to cell death in vitro (4, 14). Yet, the origin of endogenous extra-
cellular NAD
⫹
is still a matter of debate, in which both lytic and
nonlytic processes can be envisioned (25–27). Experiments re-
ported herein provide the first evidence that a significant amount of
NAD
⫹
is present in exudates during the early phase of the inflam-
matory response. Inflammation induced by polyacrylamide beads
is Ag free and thus reflects tissue reaction to a foreign bioincom-
patible material. NAD
⫹
release precedes massive neutrophil influx
into the inflammatory site. Hence, NAD
⫹
release may not be con-
ditioned by massive infiltration of neutrophils. Oxidative stress
induced by activated neutrophils, as well as hypoxia resulting from
the pressure of the Biogel mass under the skin, may contribute to
the liberation of NAD
⫹
by neighboring cells through a nonlytic
process. Importantly, the high local NAD
⫹
concentration in exu-
dates, which can reach ⬎10
M, has no effect on the plasma
NAD
⫹
level. Local NAD
⫹
release at an inflammatory site is thus
unlikely to have a global effect on T cell populations in the whole
body. Yet, NAD
⫹
concentrations in the range of 3
M were found
in the plasma of CD38
⫺/⫺
mice vs ⬍0.3
M in their wild-type
counterparts. Whether such a high concentration reflects the phys-
iological level in these mice or results from NAD
⫹
released in the
absence of the major NAD
⫹
-hydrolase CD38 during blood prep-
aration remains questionable. Micromolar NAD
⫹
concentrations
are sufficient to induce T cell death in vitro (4) and a NAD
⫹
con-
centration in exudates is sufficient to induce detectable NICD in
vitro. Yet, nonmanipulated CD38
⫺/⫺
mice have T cell numbers
and subsets similar to those in wild-type animals. It seems thus
likely that the high NAD
⫹
plasma level in CD38
⫺/⫺
animals cor-
responds to an experimental artifact. More importantly, NAD
⫹
released at a local inflammatory site induces a significant T cell
depletion in the draining but not in the nondraining nodes from
wild-type or highly sensitive CD38
⫺/⫺
BALB/c mice developing
inflammation to Biogel. This depletion is accompanied by CD62L
shedding on a significant fraction of remaining T cells, an early
event associated with P2X7 activation (18). This, in combination
with T cell resistance in ART2
⫺/⫺
mice, despite NAD
⫹
release at
the inflammatory site, provides the first evidence that NICD can
occur under physiopathological situations in vivo.
Consistently, injection of NAD
⫹
, the ART2 substrate, into nor-
mal mice expressing both ART2 and P2X7 induces massive T cell
depletion detectable within a few hours following injection. In
support of our findings, accumulation of apoptotic T cells in the
liver has been described in the C57BL/6 mouse strain within 12 h
after NAD
⫹
injection (7), although these mice harbor a partially
deficient P2X7 receptor (22). In our experiments, the depletion is
aggravated particularly in CD38
⫺/⫺
mice where it affects ⬎80% of
the T cell population in peripheral lymphoid tissues. No effect of
treatment on the thymus was observed (data not shown), in agree-
ment with the absence of ART2 on thymocytes (38). The high
sensitivity of CD38
⫺/⫺
vs wild-type BALB/c mice to the delete-
rious effect of NAD
⫹
is consistent with our previous observation
indicating that the CD38 NADase, mainly expressed on B lym-
phocytes, controls the level of ART2-mediated ADP-ribosylation
of T cell surface proteins and, therefore, T cell apoptosis in un-
fractionated peripheral lymphoid populations (28). The resistance
of T cells from ART2
⫺/⫺
BALB/c mice clearly establishes that T
cell depletion after NAD
⫹
treatment results from ART2- and
therefore P2X7-dependent NICD.
The dramatic effect of NAD
⫹
injection in vivo not only affects
the number of T lymphocytes in peripheral organs from CD38
⫺/⫺
BALB/c mice but also profoundly influences the distribution of T
cell subpopulations. The fraction of CD44
high
, CD62L
low
and
CD69
high
T cells is strongly enlarged, suggesting that cells resis-
tant to NICD in vivo are activated/memory T cells. Three argu-
ments support this conclusion. First, T cell activation leads to the
shedding of ART2 by the TACE metalloproteinase and renders
cells resistant to NICD (37, 38). Evidently, most of the CD4
⫹
and
CD8
⫹
T cells present in lymph nodes from NAD
⫹
-treated
CD38
⫺/⫺
BALB/c mice do not express ART2.2 on their surface.
Then, most of the CD4
⫹
and CD8
⫹
cells from NAD
⫹
-treated mice
have a low P2X7 density on their surface compared with controls.
We directly show that T cell activation induces a down-modulation
of P2X7 expression and resistance to NICD. This is in agreement
with the low ability of CD44
high
activated/memory CD4
⫹
T cells
to open the P2X7 large pore permeable to ethidium bromide in the
presence of ATP (39). Finally, treatment with NAD
⫹
at the time of
priming with SRBC inhibits the primary response in CD38
⫺/⫺
BALB/c mice, but treatment on day 4 after priming has no effect
or even stimulates the subsequent response to antigenic challenge
(Fig. 8). This demonstrates that primed T cells escape the delete-
rious effect of NAD
⫹
administration. Altogether, these results
demonstrate for the first time that NAD
⫹
can modulate the pool of
naive peripheral T cells in vivo, suggesting that endogenous
sources of NAD
⫹
may exert a similar effect.
One important aspect of ART2 biology concerns its role in the
regulation of the immune response. Previous experiments have
FIGURE 8. Effect of NAD
ⴙ
treatment on primary and secondary re-
sponses to SRBC. A, To determine the effect of NAD
⫹
on the primary
response, CD38
⫺/⫺
, ART2
⫺/⫺
, and wild-type BALB/c mice were primed
with 0.2 ml of a 10% SRBC suspension in PBS i.p. One-half of them
received 10 mg of NAD
⫹
in PBS or 0.2 ml of PBS i.v. at the same time as
the SRBC suspension. Agglutination titers were measured on day 6. Each
value represents the mean of six to eight individual mice from two inde-
pendent experiments. B, To determine the effect of NAD
⫹
on the secondary
response, mice were primed with SRBC on day 0. For each line, a group
of six mice received 10 mg of NAD
⫹
i.v. on day 0, another group on day
4, and the last group received NAD
⫹
on day 14. Controls received PBS on
day 0. Mice were boosted with SRBC on day 14 and Ab titers were mea-
sured on day 20. ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01 compared to controls.
192 NICD IN VIVO
shown that ART2 ADP-ribosylates several surface proteins includ-
ing LFA-1, CD8, CD27, CD43, CD44, or CD45 (10 –12). This
clearly inhibits cell contact and T cell trafficking (12). ART2-me-
diated ADP-ribosylation of membrane proteins would thus have an
inhibitory effect on the expansion of the T cell response. These
conclusions were drawn from experiments performed in C57BL/6
mice, which express ART2 at a very high density on their T lym-
phocytes (9) but have an impaired P2X7 receptor (22). Conversely,
BALB/c T cells have a lower ART2 density but display a func-
tional P2X7 receptor. Our present results indicate that in the
BALB/c genetic background, CD4
⫹
cells, which are ART2
low
P2X7
high
, are more prone to NICD in vivo than CD8
⫹
cells which
are ART2
high
P2X7
low
compared to the CD4
⫹.
This suggests that
susceptibility to NICD is governed by P2X7 more than by ART2
density.
Why should NAD
⫹
set free in the inflammatory phase of an
infection kill naive T cells during the early development of the
adaptive immune response? This situation, as illustrated by our
experiments in CD38
⫺/⫺
BALB/c mice treated with NAD
⫹
at the
time of immunization, could severely impair the response. The
same experiments, however, show that the situation is different in
a normal BALB/c mouse. Priming with NAD
⫹
does not impair the
response to Ag but even leads to a weak but significant increase of
the Ab titers. This is also verified during the secondary response
when mice are treated with NAD
⫹
4 days after priming. In these
mice, naive T cells are protected from ART2-mediated P2X7
activation and NICD by CD38 expressed on B lymphocytes.
Hence, during the early phase of the immune response, while
activated T cells down-modulate ART2 and P2X7 expression
on their surface, elimination of part of the naive T cells could
give space for the expansion of the Ag-primed T cell population
and thus increase the response. Alternatively, CD4
⫹
Foxp3
⫹
regulatory T cells may be highly sensitive to NICD, as recently
suggested in C57BL/6 mice (21). Elimination of regulatory T
cells, which exert a negative effect, could contribute toward
improving the response. However, the extent to which the ef-
fects of extracellular NAD
⫹
on the development of the immune
response can be attributed to ART-mediated NICD remains
questionable. CD38 is not only a NADase but also an ADP-
ribose cyclase. Cyclic ADP-ribose plays an important role in
the response of neutrophils and professional APCs to chemo-
tactic signals (34, 40). Consequently, CD38
⫺/⫺
mice have a
diminished Ab response to T-dependent Ags (29). The reduced
response to SRBC of CD38
⫺/⫺
vs wild-type BALB/c mice (Fig.
8A) is in fair agreement with this conclusion. Combined with
NICD, a defect in Ag presentation may thus explain the dra-
matic impairment of the primary SRBC response in CD38
⫺/⫺
mice treated with a high dose of NAD
⫹
. Similarly, in CD38
⫹/⫹
BALB/c mice, NAD
⫹
injection might increase cyclic ADP-ri-
bose production and improve APC activation and trafficking,
compensating the deleterious effect of NICD or even improving
the response. The observation that NAD
⫹
administration does
not enhance the primary response in ART2
⫺/⫺
mice, however,
makes it likely that this effect is primarily due to ART2-
mediated NICD.
Taken together, our results demonstrate that NAD
⫹
is released
during the early phase of inflammation and illustrate its role in
immune regulation in vivo. They also stress that ART2 and CD38
are two important, but not necessarily opposed partners in this
regulation. Evidently, NICD is a phenomenon which can occur in
vivo. Yet further investigation is needed to define its main target
and its role in the regulation of T cell homeostasis and the control
of autoimmune diseases in normal mice.
Disclosures
The authors have no financial conflict of interest.
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