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INTRODUCTION |
Hypertonic stress (HS)1
increases cytokine expression of immune cells and enhances T cell
proliferation (1, 2). Peripheral blood mononuclear cells show increased
production of IL-1
, IL-1
, IL-8, and tumor necrosis factor-
when the cells are exposed to elevated extracellular tonicity (3).
Hypertonic stimulation augments the ability of T cells to produce IL-2,
tumor necrosis factor-
, and lymphotoxin-
(2, 4) and restores IL-2
expression in the presence of anti-inflammatory mediators (5).
Hypertonic solutions can be used to resuscitate trauma patients, who
are predisposed to septic complications, and can improve T cell
function after hemorrhagic shock, reducing the risk of sepsis after
trauma (1, 6).
The enhancing effect of HS on IL-2 expression is paralleled by robust
tyrosine phosphorylation of a number of intracellular proteins
including p38 MAPK (2). This kinase is structurally related to the
yeast protein HOG-1, which is part of the signaling system that allows
yeast cells to regulate gene transcription in response to osmotic
stress (7). In human T cells, p38 MAPK signaling is involved in the
activation of IL-2 gene expression (8). Therefore, by analogy with its
yeast counterpart, osmotic activation of the p38 MAPK may be involved
in the mechanisms whereby HS enhances IL-2 expression of T cells.
In yeast, two osmoreceptors are known to activate HOG-1. No equivalent
mammalian osmoreceptors have been identified to date, and, thus, the
mechanisms whereby T cells respond to extracellular tonicity are
unclear (7). HS results in cell shrinkage and mechanical deformation of
the cell membrane. In a number of cell types, mechanical stress
activates multiple signaling enzymes, including p38 MAPK (9, 10). Thus,
the mechanisms by which these cells detect and respond to direct
mechanical stimulation may be similar to those involved in the
recognition of HS.
Mechanical stimulation can cause the rapid release of ATP (11-13).
Once released into the extracellular environment, ATP can regulate cell
function in an autocrine/paracrine manner by interacting with P2X
and/or P2Y receptors that are expressed on the surface of virtually all
mammalian cells (13-16). Extracellular ATP and its metabolic products,
including adenosine, which acts via P1 receptors, exert a strong
influence on lymphocyte function: ATP can stimulate the proliferation
of mouse thymocytes, and ATP and adenosine can antagonize and/or
complement T cell receptor-induced signaling, apoptosis, and thymocyte
differentiation (16-18). In this study, we tested the hypothesis that
ATP release in response to osmotic stimulation could be a mechanism
whereby HS enhances IL-2 expression of T cells.
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EXPERIMENTAL PROCEDURES |
Materials--
ATP and a nonhydrolyzable ATP analog,
ATP
S, suramin, apyrase, adenosine deaminase, and
Me2SO were from Sigma, whereas
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 2',3'-dialdehyde ATP (oxidized ATP, o-ATP),
pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and
SB203580 were from Calbiochem. The Ca2+-sensitive
intracellular probe Fura-2 was obtained from Molecular Probes, Inc.
(Eugene, OR). Antibodies to CD3 were purified from the supernatant of
OKT-3 cells (clone CRL 8001) obtained from ATCC and anti-CD28
antibodies (clone CD28.2) were from BD Pharmingen (San Diego, CA).
Polyclonal goat anti-P2X7 antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), and normal horse serum,
biotinylated anti-goat antibodies, and fluorescein avidin D cell sorter
grade conjugate were from Vector Laboratories (Burlingame, CA).
Cells and Cell Stimulation--
Jurkat T cells (clone E6-1)
were obtained from the American Type Culture Collection (Manassas,
VA) and maintained in RPMI 1640 (Irvine Scientific, Santa Ana,
CA) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin
(University of California San Diego core facility, La Jolla, CA), and
10% (v/v) heat-inactivated fetal calf serum (Irvine Scientific).
Peripheral blood mononuclear cells (PBMC) were isolated from the
heparinized venous blood of healthy human volunteers as described
before (5).
Jurkat cells were stimulated by simultaneously activating the T cell
receptor-CD3 complex and CD28 co-receptor with Dynabeads (Dynal Inc.,
Lake Success, NY) coated with anti-CD3 and anti-CD28 antibodies. The
beads (107), precoated with anti-mouse IgG, were coated
with anti-CD3 and anti-CD28 by incubation with 5 µg of each antibody
at room temperature for 1 h. Then the beads were washed twice with
RPMI with 10% fetal calf serum and resuspended at 107
beads/ml, and 20 µl was added to 2 × 104 Jurkat
cells (10 beads/cell). The cells were incubated in a final volume of
200 µl for 20 h at 37 °C under tissue culture conditions, and
IL-2 in the supernatant was determined. PBMC were suspended in the RPMI
medium described above (105/ml) and stimulated under tissue
culture conditions with 0.5 µg/ml phytohemagglutinin (PHA; Abbott) in
a final volume of 200 µl for 20 h. IL-2 concentrations in the
supernatants were determined with the enzyme-linked immunosorbent assay
method described below.
Where indicated, cells were pretreated for 1 h at 37 °C with
different agents including P1 and P2 receptor antagonists or the p38
MAPK inhibitor SB203580 (19). Cells were subjected to HS by adding
appropriate volumes of RPMI containing 1 M NaCl. All
materials and compounds used in these experiments were sterile and
endotoxin-free.
ATP Release--
ATP release from Jurkat cells was monitored in
real time with cells suspended in luciferase reagent. Cells were
brought to a density of 5 × 105/ml in RPMI without
fetal calf serum, and 25 µl/well was transferred to a 96-well
luminometer plate. Luciferase reagent in RPMI (25 µl/well) was then
added (ATP Bioluminescence Assay Kit CLS II; Roche Diagnostics GmbH,
Mannheim, Germany). The plate was placed in a temperature-controlled
luminometer (Luminoskan; Labsystems, Helsinki, Finland), and sequential
readings were taken as indicated. For quantitative ATP measurements,
cells were preincubated for 3 h at 37 °C and stimulated with
hypertonic saline for different periods. The cells were then placed on
ice, and ATP concentrations in the supernatants were determined.
IL-2 Expression--
IL-2 released into the supernatants after
20 h was measured with an enzyme-linked immunosorbent assay using
monoclonal mouse anti-human IL-2 as a primary antibody (clone 5355.111)
and biotinylated goat anti-human IL-2 as a secondary antibody (both
from R&D Systems Inc., Minneapolis, MN), recombinant human IL-2 as a
standard (Genzyme Diagnostics, Cambridge, MA), and horseradish
peroxidase-conjugated streptavidin (Zymed Laboratories
Inc., San Francisco, CA). The enzyme-linked immunosorbent assay
was performed according to the recommendations provided by R&D Systems.
p38 MAPK Expression and Activation--
To measure p38 MAPK
activation, Jurkat cells (106 cells/sample) stimulated in
the different experiments were placed on ice, centrifuged, resuspended
in 100 µl of ice-cold SDS sample buffer containing 100 mM
dithiothreitol, and lysed by boiling for 5 min. The cell lysates were
separated by SDS-PAGE electrophoresis using 8-16% Tris/glycine
polyacrylamide gradient gels (Novex, San Diego, CA). Lysed proteins
were transferred to polyvinylidene difluoride membranes (Immobilon-P;
Millipore Corp., Bedford, MA), and the membranes were subjected to
immunoblotting with phospho-specific antibodies that recognize the
phosphorylated (on Thr180/Tyr182), and thereby
activated, form of p38 MAPK (New England Biolabs, Beverly, MA).
Antibodies recognizing both active and inactive p38 MAPK were from
Santa Cruz Biotechnology, and secondary antibody conjugates as well as
the ECL assay kit were from Amersham Biosciences.
Intracellular Ca2+ Measurements--
Intracellular
Ca2+ levels were determined as described previously (20)
using the fluorescent Ca2+ probe Fura-2 and a
spectrofluorimeter (Kontron Instruments, Zurich, Switzerland). In
experiments, where extracellular Ca2+ was chelated, EGTA
(Calbiochem) was added at a final concentration of 10 mM.
P2X7 Receptor Expression--
Expression of the P2X7 receptor in
Jurkat cells was determined by immunoblotting of cell membrane
fractions and by immunofluorescence staining. Cells were washed in
ice-cold phosphate-buffered saline, resuspended in 5 ml of
homogenization buffer (30 mM HEPES, 5 mM MgCl2, 1 mM EGTA, and 2 mM
dithiothreitol, pH 7.5), homogenized with a sonicator, and centrifuged
at 300 × g for 5 min at 4 °C. The supernatant was
centrifuged at 5,000 × g for 10 min at 4 °C, resulting in a pellet containing a crude membrane preparation. Purified plasma membrane preparations were obtained by density gradient
centrifugation of the cell homogenate using 30% Percoll at 64,000 × g for 30 min. The crude and purified membrane
preparations were boiled in SDS sample buffer and subjected to
SDS-PAGE and immunoblotting with goat anti-P2X7 antibodies (Santa Cruz Biotechnology).
For immunostaining, cells were attached to microscope slides by
cytocentrifugation, fixed with chilled acetone for 10 s, and dried. Then the cells were blocked with 10% normal horse serum in
phosphate-buffered saline for 20 min and incubated with 20 µg/ml goat
anti-P2X7 antibody in 10% horse serum for 1 h. The cells were
washed with phosphate-buffered saline, incubated for 10 min with
biotinylated horse anti-goat IgG antibody in 10% horse serum, washed
with phosphate-buffered saline, and incubated for another 10 min with
fluorescein avidin D cell sorter grade conjugate according to
the supplier's recommendations. The cells were washed five times with
phosphate-buffered saline, mounted with Crystal/Mount (Biomeda Corp.,
Foster City, CA), and examined with a fluorescence microscope (Leica,
Wetzlar, Germany). Fluorescence and modulation contrast images were
acquired of cells that were stained with anti-P2X7 and of control cells
that were treated identically to the antibody-stained cells, except
that the primary anti-P2X7 antibody was omitted.
Statistical Analyses--
Unless otherwise indicated, data are
presented as mean ± S.D. Sets of data were compared with
Student's t test, using p < 0.05 as the
level of significance.
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RESULTS |
Mechanical Stimulation and HS Cause Rapid ATP Release--
We
first investigated whether mechanical stimulation causes ATP release
from Jurkat cells. Cells were suspended in RPMI containing luciferase reagent, and ATP release was monitored on-line with a
luminometer. Initially high luminescence readings, indicative of ATP
release due to cell handling, gradually decreased, reaching base-line
levels within 150 min (Fig.
1A, left). When the
cells were mechanically stimulated by gentle aspiration with a pipette four times, the luminescence signal rapidly peaked within 1 min and
returned toward base-line levels by 100 min. These results show that
mechanical stimulation causes the release of ATP into the extracellular
space. The steady decline of luminescence readings after mechanical
stimulation indicates that ATP release ceases shortly after the
stimulus and that released ATP is hydrolyzed, presumably by
ectoapyrases, ecto-ATPases, and/or ecto-5'-nucleotidases expressed on
the Jurkat cell surface (21).

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Fig. 1.
HS causes rapid ATP release and enhances IL-2
expression. A, ATP release from Jurkat T cells
suspended in luciferase reagent was monitored in a plate luminometer.
Cells (1.25 × 104) suspended in RPMI containing
luciferase reagent were pipetted into the wells of a 96-well plate and
left undisturbed until luminescence readings returned to base-line
levels (shown in left panel). At time point 0 min, the cells were mechanically agitated by gently pipetting the cell
suspensions up and down four times (left) or exposed to
osmotic stress by adding equal volumes of RPMI containing additional
NaCl to increase the final tonicity of the culture medium by 0, 40, 80, or 160 mM (right). The results shown are
representative of three experiments performed on different days, and
values (relative luminescence units (RLU)) are expressed as
the mean ± S.D. of triplicate determinations. B, ATP
released into the culture supernatant was determined with a
commercially available ATP bioluminescence assay kit. Jurkat T cells
(5 × 106/ml) were allowed to rest for 3 h at
37 °C before they were osmotically stimulated with 100 mM HS (left) or the indicated hypertonicity
levels (right) in the presence of 200 µM
suramin to prevent ATP degradation. ATP concentrations in the culture
supernatants of quadruplicate samples were determined at the indicated
time points (left) or after 5 min (right). The
results shown consist of the average ± S.D. of three experiments
that were performed on different days. The asterisks
indicate statistically significant differences from isotonic control
values (*, p = 0.001; **, p < 0.001).
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Because HS causes rapid cell shrinkage, we hypothesized that the
associated mechanical forces could trigger ATP release from T cells. We
tested this possibility by monitoring ATP release in response to
increasing levels of HS. The addition of hypertonic solutions rapidly
released ATP in a concentration-dependent fashion (Fig.
1A, right). Peak luminescence signals reached a
maximum within 4 min and gradually decreased to base-line readings with kinetics similar to those observed with mechanical agitation. The peak
ATP signal in response to stimulation with 80 mM
hypertonicity was more than 3 times as high as that observed in
response to 40 mM hypertonicity. ATP release was less
pronounced in response to 160 mM, suggesting that higher
levels of hypertonicity may exert suppressive effects on ATP release.
The relation between HS levels and ATP release was further examined by
measuring ATP release using an approach similar to that described by
Yegutkin et al. (12), who prevented the degradation of
extracellular ATP with suramin, a drug that blocks P2 receptors and
also inhibits ecto-5'-nucleotidases. These experiments confirmed that
exposure to HS causes a fast and dose-dependent increase in
extracellular ATP. The addition of an equivalent volume of isotonic
control solution caused a considerably smaller increase in
extracellular ATP, which is likely the result of mechanical perturbations caused by adding this solution (Fig. 1B,
left). ATP release increased with hypertonicity levels
peaking at 80 mM HS (Fig. 1B,
right).
HS and Extracellular ATP Co-stimulate IL-2 Expression--
The
similarities between ATP release and other T cell responses to HS
suggested that ATP release could be involved in the mechanisms whereby
HS enhances IL-2 expression. To test this possibility, Jurkat cells
were stimulated with antibodies to CD3 and CD28 in the presence of
increasing levels of HS or exogenous ATP, and IL-2 production was
measured 20 h later. HS at concentrations up to 60 mM
increased IL-2 expression of Jurkat cells and isolated PBMC, whereas
higher levels of HS suppressed IL-2 production (Fig. 2A). Similar to HS, the
addition of exogenous ATP significantly increased IL-2 expression of
Jurkat cells in a concentration-dependent fashion (Fig.
2B, left). ATP concentrations of 10-40
µM significantly increased IL-2 expression. As with
increasing levels of HS, higher concentrations of ATP progressively
reduced the enhancement of IL-2 expression. The nonhydrolyzable ATP
analog ATP
S also enhanced IL-2 expression, doubling IL-2 expression
at concentrations of
20 µM (Fig. 2B,
left). In contrast to the results with ATP and HS, we
observed no bimodal effect at higher concentrations of ATP
S. UTP at
concentrations between 0.02 and 1 µM also enhanced IL-2
expression, whereas ADP and adenosine (ADO) had no
discernible effects (Fig. 2B, right). These data
demonstrate that certain nucleotides, but not ADP and adenosine, can
co-stimulate IL-2 expression, supporting the notion that activation of
specific nucleotide receptors may contribute to the regulation of T
cell function by HS.

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Fig. 2.
HS and ATP enhance IL-2 expression.
Jurkat cells (A, left) and isolated human PBMC
(A, right) were stimulated in the presence of
increasing HS concentrations with anti-CD3/CD28 antibodies or PHA,
respectively, and IL-2 expression was determined after 20 h.
B, the effects of exogenous ATP, ATP S (left),
UTP, ADP, and adenosine (ADO; right) on IL-2
expression of anti-CD3/CD28-stimulated Jurkat cells was determined. The
results shown are representative of three individual experiments, and
values are expressed as mean ± S.D. of triplicate determinations;
asterisks indicate statistically significant differences
from control values in the absence of HS or nucleotides (*,
p < 0.05; **, p 0.01).
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HS and ATP Cause Ca2+ Mobilization--
Extracellular
nucleotides, such as ATP, act via plasma membrane P2Y and P2X receptors
(14, 16, 18). Activation of both receptor types results in
intracellular Ca2+ mobilization. To test whether exposure
of Jurkat cells to HS or ATP could elicit intracellular
Ca2+ mobilization, we loaded Jurkat cells with the
Ca2+ probe Fura-2 and assessed [Ca2+] in
response to HS or the addition of ATP. HS rapidly and
concentration-dependently increased intracellular
[Ca2+] in Jurkat cells (Fig.
3A, left). This
response was blunted when extracellular ATP was hydrolyzed with apyrase
or when extracellular Ca2+ was bound with EGTA (Fig.
3A, right). The peak Ca2+
concentrations corresponded to the applied levels of HS and were consistently reduced by apyrase (Fig. 3B). Exogenous ATP and
ATP
S mobilized Ca2+ over a similar time course (Fig.
3C). Whereas 0.5-1.0 mM ATP was required to
mobilize calcium, ~100-fold lower concentrations of ATP
S increased
intracellular [Ca2+], suggesting that much of the added
ATP may be hydrolyzed before it can activate P2 receptors on the T cell
membrane. Together, these findings suggest that HS-promoted ATP release
can trigger secondary signaling events, such as Ca2+
mobilization via activation of purinergic receptors, which may be
involved in the up-regulation of IL-2 expression by HS.

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Fig. 3.
HS and ATP mobilize intracellular
Ca2+. Jurkat T cells were loaded with the
Ca2+ probe Fura-2, exposed to the indicated levels of HS
(A, left panel) or to HS (300 mM) in the presence or absence of apyrase or EGTA
(A, right panel), and Ca2+
mobilization was recorded. Peak intracellular Ca2+ levels
in the presence or absence of apyrase were plotted versus
the corresponding HS levels (B). For comparison,
Ca2+ mobilization in response to stimulation with the
indicated concentrations of ATP or ATP S was determined
(C). Data shown are representative of three different
experiments with similar results.
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HS Enhances IL-2 Expression via Released ATP--
As a further
test of the idea that ATP released in response to HS augments the
ability of CD3/CD28-stimulated T cells to express IL-2, we exposed
CD3/CD28-stimulated Jurkat cells to HS in the presence of apyrase, an
enzyme that scavenges extracellular ATP (22). Apyrase
concentration-dependently reduced the co-stimulatory effects of HS (Fig. 4A). At a
concentration of 20 units/ml, apyrase almost completely prevented the
enhancement of IL-2 expression by increasing levels of HS (Fig.
4B). These data are consistent with the idea that ATP
released into the extracellular space plays a critical role in the
events that lead to enhanced IL-2 expression in the presence of HS. As
a second approach to examine the role of ATP release in the enhancement
of IL-2 expression, we used the P2 receptor antagonist, suramin, which
nonselectively blocks P2 receptors and, in addition, as noted above,
inhibits ecto-ATPases (14). Suramin significantly reduced the enhancing
effects of HS on IL-2 production (Fig. 4C). ATP can be
hydrolyzed to adenosine by ectoenzymes (17), and we investigated
whether adenosine might be involved in the enhancing effects of HS on
IL-2 production. The addition of adenosine deaminase to hydrolyze
adenosine did not alter the effect of HS on IL-2 expression (Fig.
4D). This suggests that ATP but not adenosine is responsible
for the enhancing effects of HS.

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Fig. 4.
Extracellular ATP is required for the
enhancement of IL-2 expression by HS. Jurkat cells were stimulated
with bead-bound anti-CD3/CD28 antibodies in the presence of the
indicated HS levels and of increasing concentrations of apyrase
(A) or of 20 units/ml apyrase (B) to break down
released ATP, in the presence (empty symbols) or
absence (filled symbols) of 200 µM
suramin to block P2 receptors (C) or in the presence
(empty symbols) or absence (filled
symbols) of 1 unit/ml adenosine deaminase (ADA).
IL-2 production was determined as described above. Results are
representative of three individual experiments, and data are means ± S.D. of triplicate determinations. Asterisks indicate
significant differences between treated versus untreated
samples (B and C) and HS versus
isotonic controls (A) (*, p < 0.05; **,
p 0.01).
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Role of P2X7 Receptors--
Stimulation of P2X7 receptors has been
reported to increase intracellular [Ca2+] and to
synergize T cell proliferation in response to mitogens, such as PHA
(18). We used two approaches, immunoblotting of membranes (Fig.
5A) and immune fluorescence
analysis of cells (Fig. 5B), and found that Jurkat T cells
express P2X7 receptors. In addition, we tested the role of P2X7 in the
response of T cells to HS using the P2 receptor antagonists KN-62,
o-ATP, and PPADS, all of which antagonize P2X7 receptors (14). Jurkat T
cells or isolated PBMC were treated with increasing concentrations of these antagonists for 20 min, exposed to 40 mM HS, and
stimulated with CD3/CD28 or PHA, respectively. With IC50
values of about 0.1 µM, KN-62 was the most potent of the
three compounds in reducing the effect of HS on IL-2 production (Fig.
5C). PPADS and o-ATP also reduced IL-2 expression, although
higher antagonist concentrations were needed; both PPADS and o-ATP
suppressed IL-2 expression of PBMC beyond the IL-2 expression level
reached under isotonic conditions (dashed line).
PBMC preparations consist not only of T cells but also of B cells and
monocytes; the antagonists may affect P2 receptors in these other cells
that serve as accessory cells to T cells. The inhibitory effect by
o-ATP was observed with both Jurkat and peripheral T cells, suggesting
that o-ATP may not only affect the P2X7 receptor but also perhaps other
P2 receptors that influence T cell activation under normal, isotonic
conditions.

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Fig. 5.
P2X7 receptor expression and action in Jurkat
cells and PBMC. Jurkat cells express P2X7 as shown by
immunoblotting of crude membranes (MB) and purified
membranes (PM) of Jurkat cells (A) and in the
fluorescence image of Jurkat cells stained with P2X7 antibodies
(B, upper panels) or control cells
that were stained without P2X7 antibodies (B,
lower panel). Jurkat cells (C,
left) or isolated human PBMC (C,
right) were pretreated for 20 min with the indicated
concentration of the P2X7 antagonist PPADS, o-ATP, or KN-62,
stimulated with 40 mM HS and anti-CD3/CD28 or PHA,
respectively. IL-2 production was determined as in Fig. 2 and is
expressed as a percentage of the IL-2 production of control cells that
were stimulated in the absence of HS.
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ATP, Like HS, Activates p38 MAPK--
HS is known to activate p38
MAPK in T cells (2). Because p38 MAPK signaling targets several nuclear
factors that may be responsible for T cell gene regulation, we tested
whether p38 MAPK contributes to the mechanisms through which HS
enhances IL-2 expression. We thus compared the roles of p38 MAPK
signaling in response to stimulation with ATP or HS. HS levels of 20, 40, and 100 mM activated p38 MAPK in a concentration- and
time-dependent fashion (Fig.
6A). p38 MAPK activation was
detectable at 40 and 100 mM hypertonicity by 3 min, but the
highest activation levels were observed 30-60 min after stimulation
with HS. Stimulation of T cells with ATP caused a less pronounced
activation of p38 MAPK with a peak at 1 min (Fig. 6B). ATP
concentrations between 0.01 and 1 µM were most effective
in the stimulation of p38 MAPK activation (Fig. 6C). These
data show that extracellular ATP can trigger p38 MAPK activation and
that ATP release in response to hypertonic stimulation of T cells may
contribute to HS-promoted activation of p38 MAPK. A reason for the
different kinetics and amplitudes of p38 MAPK stimulation by HS and ATP
may be the prolonged nature of hypertonic stimulation compared with
stimulation with ATP.

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Fig. 6.
HS and ATP activate p38 MAPK in Jurkat
cells. Timing of p38 MAPK activation by HS (A) and ATP
(B) and the dose response to ATP stimulation were determined
by stimulating Jurkat cells with the indicated levels of HS or ATP for
different periods or for 5 min (C). p38 MAPK activation was
determined by immunoblotting with phosphospecific anti-p38 MAPK
antibodies (upper lanes). To control for equal
protein loading, membranes were reprobed with antibodies recognizing
active and inactive p38 MAPK (lower lanes). Band
intensities were analyzed, and ratios between activated and total p38
MAPK were used to calculate p38 MAPK activation. In B and
C, cells were stimulated with 100 mM HS as a
positive controls. The data shown are representative of three
experiments with similar results.
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p38 MAPK Is an Important Element in the Mechanism whereby HS
Enhances IL-2 Expression--
The findings above suggest that p38 MAPK
signaling may contribute to the enhancement of IL-2 expression by HS
and ATP. We thus inhibited p38 MAPK with the inhibitor SB203580 and
investigated whether this would prevent the enhancing effects of HS on
CD3/CD28-stimulated IL-2 expression. SB203580
concentration-dependently reduced the enhancement of IL-2
expression by 40 mM hypertonicity. Increasing concentrations of SB203580 also reduced IL-2 expression of cells stimulated in the absence of HS (Fig. 7).
The latter finding corresponds to earlier reports that suggested a role
of p38 MAPK in the activation of IL-2 expression of Jurkat cells (8).
SB203580 completely abrogated the enhancement of IL-2 expression by a
wide range of HS conditions (Fig. 7). These results imply that p38 MAPK
plays a major role in the enhancement of IL-2 expression by HS.

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Fig. 7.
Osmotic regulation of IL-2 expression
involves ATP release and p38 MAPK. Jurkat T cells were treated for
1 h at 37 °C with either the indicated doses of the p38 MAPK
inhibitor SB203580 (left), 20 µM SB203580, or
Me2SO vehicle only (no SB203580) (right). The
cells were then stimulated via CD3/CD28 in the absence or presence of
HS as indicated, and IL-2 production was determined. Data are
representative of three different experiments and expressed as
mean ± S.D. (*, p < 0.03; **, p < 0.001, n = 3).
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DISCUSSION |
Under normal physiological conditions, plasma tonicity can vary
significantly as a consequence of water loss and salt uptake. Plasma
tonicity is affected by age (23), climate, and seasonal changes (24)
and by activities such as water/salt uptake and physical exercise (25,
26), the latter of which can affect the ratio of T cell subsets in the
circulation (25). Whereas these external influences can alter plasma
tonicity, even more extreme differences in extracellular tonicity occur
within the organs of the gastrointestinal and urinary tract. It is
possible that elevated tonicity could increase the level of vigilance
of the immune cells patrolling these organs.
Aside from HS, other conditions leading to cellular distress can result
in ATP release (e.g. tissue injury or hypoxia during hemorrhagic shock) (27). ATP and its metabolites may allow the host to
better cope with injury by increasing T cell function. Indeed,
administration of ATP-MgCl2 or inhibition of adenosine kinase has been shown to improve survival after hemorrhagic shock and
sepsis and to protect lungs from ischemia reperfusion injury (27-29).
Because of its protective effects, the ATP metabolite, adenosine, has
been termed the "retaliatory metabolite in ischemia-reperfusion" (27).
In trauma patients, hypertonic solutions can be used to rapidly restore
lost blood volume (6). The infusion of these solutions transiently
increases plasma tonicity to levels that we report here to cause
substantial ATP release from T cells. As observed with
ATP-MgCl2 administration, hypertonic solutions can improve the outcome after hemorrhagic shock and sepsis in both animal models
and patients (1, 6). The present study suggests that ATP release in
response to hypertonic solutions could mediate these beneficial
in vivo effects by enhancing the function of T cells.
It has long been recognized that cells release ATP under pathological
conditions that involve hypoxia, cell damage, loss of cell viability,
and cytolysis (14, 15). More recently, it has been noted that healthy
cells release ATP under normal physiological conditions. Mechanical and
osmotic stimulation have been shown to cause ATP release from several
different cell types including endothelial and epithelial cells,
fibroblasts, hepatocytes, and vascular smooth muscle cells
(e.g. Refs. 12, 13, 30, and 31). There are at least three
possible mechanisms for the regulated release of nucleotides under
physiological conditions: exocytosis of ATP-filled vesicles, conductive
transport through membrane ion channels, and facilitated diffusion by
nucleotide-specific transporters. However, work to date has not
precisely resolved which mechanism is operative (22, 30).
The present study demonstrates for the first time that mechanical
agitation and exposure to HS stimulate Jurkat T lymphocytes to release
ATP in a manner that correlates with the level of hypertonicity to
which the cells are exposed (Fig. 1). Similar ATP release patterns have
been reported in response to direct mechanical stimulation of
endothelial and vascular smooth muscle cells (13, 32). Exposure of
mammalian cells to HS causes rapid cell shrinkage, which is accompanied
by membrane deformation. Modest mechanical forces of hemodynamic origin
and forces associated with increased perfusion flow rates have been
shown to trigger ATP release from vascular smooth muscle and
endothelial cells (15, 32, 33). Mechanical and hypertonic stimulation
of T cells resulted in a rapid ATP release followed by a decrease
toward base-line levels (Fig. 1). This decrease of extracellular ATP is
presumed to occur via ATP hydrolysis by ecto-ATPases and
ecto-5'-nucleotidases, such as CD39 and CD73, which may be expressed on
the T cell membrane (18, 21). Other pathways for metabolism of
extracellular nucleotides have been identified recently
(e.g. Ref. 34).
As with HS, the addition of exogenous ATP, its nonhydrolyzable analog
ATP
S, and UTP significantly increased the ability of Jurkat cells to
produce IL-2, and the addition of apyrase blunted this response (Figs.
2 and 4). The results demonstrate that ATP release in response to
osmotic stimulation can enhance IL-2 expression in an
autocrine/paracrine fashion that probably involves P2 receptors expressed on the T cell surface. The ATP product adenosine
(ADO) did not influence IL-2 expression (Fig.
2B), and adenosine deaminase, a scavenger of adenosine, did
not reduce the enhancing effect of HS on IL-2 production (Fig.
4D), suggesting that adenosine and the adenosine or P1
receptors are not involved in the regulation of T cell function by
HS.
Based on their molecular structures, P2 receptors are divided into two
subfamilies: the G protein-coupled heptahelical P2Y receptors and P2X
receptors that are ligand-gated ion channels. Of the mammalian P2
receptors that have been cloned to date (14), only P2X-like receptors
(P2X1, P2X4, and P2X7) seem to be expressed by human T lymphocytes
(18). We found that Jurkat cells express P2X7 and that P2X7-specific
antagonists can attenuate the enhancing effect of HS on IL-2 expression
(Fig. 5). These observations suggest that P2X7 may play an important
role in the response of T cells to HS.
HS triggered robust Ca2+ mobilization at HS levels that
increased extracellular ATP concentrations to ~2 µM
(Fig. 1B). In comparison, much higher concentrations of
exogenous ATP were required for equivalent increases of intracellular
Ca2+ levels (Fig. 3) or p38 MAPK activation (Fig. 6). This
discrepancy could be due to differences of the ATP concentrations in
proximity to the T cell membrane compared with the bulk media.
Extracellular nucleotides can be hydrolyzed by ecto-ATPases and
ecto-5'-nucleotidases that exist on the plasma membrane of cells (21,
34); our data (Fig. 1) suggest the presence of such enzymes on Jurkat
cells. ATP released in response to hypertonic stimulation is likely to achieve its highest concentrations in close proximity to the plasma membrane (i.e. adjacent to P2 receptors) (35). This
explanation is consistent with our observation that nonhydrolyzable
ATP
S was about 50 times as effective as ATP in triggering
Ca2+ mobilization in Jurkat cells. Mechanical stimulation
of other cell types causes ATP release and the propagation of
Ca2+ waves along monolayers, indicating that ATP-induced
Ca2+ signaling can serve as a means of communication
between neighboring cells (36). Our data suggest that HS-induced ATP
release may also serve as messenger between osmotically stressed cells.
p38 MAPK is activated by cellular stresses, inflammatory cytokines,
lipopolysaccharide, and G protein-coupled receptors (37, 38). Activated
p38 MAPK in turn mediates cytokine production and other stress
responses (19, 38). p38 MAPK is also activated by mechanical
stimulation in a number of cell types (e.g. Refs. 39 and
40). Mechanical stimulation of vascular smooth muscle cells recently
has been shown to activate p38 MAPK through a mechanism that involves
ATP release and a feedback mechanism involving extracellular ATP and
adenosine (32). Our data suggest that a similar feedback mechanism may
be involved in the response of Jurkat T cells to HS (Fig. 6).
Inhibition of p38 MAPK with SB203580 prevented the enhancement of IL-2
expression by HS, consistent with the idea that p38 MAPK signaling
plays a critical role in the response of T cells to HS.
Overall, our results suggest a regulatory pathway where HS causes the
release of ATP. ATP in turn activates P2 receptors that increase
Ca2+ influx and the activation of p38 MAPK. Finally, p38
MAPK exerts a co-stimulatory signal that enhances IL-2 expression of
CD3/CD28-stimulated T cells, thereby enhancing T cell function (Fig.
8). Existence of this pathway identifies
new targets for the modulation of T cell function.

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Fig. 8.
Proposed mechanism of hypertonic regulation
of IL-2 expression. We propose that HS enhances IL-2 expression of
T cells via osmotic cell shrinkage, membrane deformation, ATP release
into the extracellular space, the activation of P2X7 by ATP, and
subsequent Ca2+ mobilization and p38 MAPK activation. These
signals then increase IL-2 gene transcription and expression.
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