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Modulation of human t cell responses by nitric oxide and its derivative s-nitrosoglutathione.

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1414
ARTHRITIS & RHEUMATISM Volume 36
Number 10, October 1993, pp 14141422
0 1913, American College of Rheumatology
MODULATION OF HUMAN T CELL RESPONSES BY
NITRIC OXIDE AND ITS DERIVATIVE,
5’-NITROSOGLUTATHIONE
PARVIN F. MERRYMAN, ROBERT M. CLANCY, XIAO YUEN HE, and STEVEN B. ABRAMSON
Objective. To examine the effects of nitric oxide
(NO) and its more stable derivative, S-nitrosoglutathione (SNO-GSH), on the response of activated T
lymphocytes.
Methods. The effects of NO and SNO-GSH on
DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor expression, and cGMP accumulation were determined in phytohemagglutinin-activated peripheral blood
mononuclear cells (PBMC) and spleen T cells.
Results. Nitric oxide (half-life [T,,,] <15 seconds)
did not inhibit T cell proliferation. However, the derivative SNO-GSH (25 ErM) (TI,, >2 hours) inhibited DNA
synthesis by a mean 2 SD of 65 2 19.6% (P< 0.001) in
PBMC and 75 f 15% (P < 0.001) in spleen cells.
Macrophage depletion of PBMC did not abrogate the
inhibition. SNO-GSH had no effect on IL-2 production
or IL-2 receptor expression. NO (25 ptf) increased the
cGMP content of PBMC (0.65 f 0.15 pmoles/106 cells;
P < 0.04), as did SNO-GSH (25 ptf) in both PBMC (3.8
2 1; P < 0.001) and spleen T cells (5.2 f 1.2; P <
0.001). Methylene blue and hemoglobin, which are NO
inhibitors, inhibited SNO-GSH-induced cGMP accumulation (P< 0.001).
Conclusion. SNO-GSH inhibits T cell DNA synthesis independently of IL-2 production and in associa____
From the Department of Rheumatology and Molecular
Medicine, Hospital for Joint Diseases, and the Department of
Medicine, Division of Rheumatology, New York University School
of Medicine, New York, NY.
Parvin F. Merryman, PhD; Robert M. Clancy, PhD; Xiao
Yuen He, MS; Steven B. Abramson, MD.
Address reprint requests to Parvin F. Merryman, PhD,
Department of Rheumatology, Hospital for Joint Diseases, 301 East
17th Street, New York, NY 10003.
Submitted for publication January 7, 1993; accepted in
revised form March 22, 1993.
tion with cGMP accumulation via a NO-dependent
mechanism. We suggest that NO and its S-nitrosothiol
derivatives may act as endogenous inhibitors of T cellmediated inflammation.
Nitric oxide (NO), an endothelium-derived relaxation factor (EDRF), is synthesized via arginine
oxidation by a family of nitric oxide synthases (NOS)
in a variety of tissues (1). A gaseous molecule, NO is
labile and in the presence of oxygen, is rapidly metabolized to nitrates and nitrites (2,3). However, NO can
also react with reduced thiols to form S-nitrosothiol
derivatives, which are more long-lived EDRFs (4).
Both NO ( 5 ) and its S-nitrosothiol derivatives (6)
activate soluble guanylate cyclase, thus increasing
cellular cGMP levels, and through this mechanism
may mediate effects upon platelets and smooth muscle
cells (6-10).
Although originally recognized as a product of
vascular endothelium, NO is synthesized by, and is
biologically active in, a variety of other cell types such
as macrophages (2,11,12), cerebral neurons (13), pancreatic B cells (14), synoviocytes (15), and neutrophils
(16,17). Isomeric forms of NOS have been cloned from
bovine, rat, mouse, and human tissues (18-22). Because of its capacity to react with heme and non-heme
iron as well as sulfhydryl groups of diverse proteins,
nitric oxide exerts a broad range of effects on cells. In
macrophages, NO exhibits antimicrobial effects and
contributes to cytotoxic activity against tumor cells
(23-25). In human neutrophils, nitric oxide inhibits
superoxide anion production via direct action on a membrane component of the NADPH-oxidase (26), and it has
been implicated in the killing of bacteria (27).
There is increasing evidence that nitric oxide
NO AND SNO-GSH MODULATE T CELL RESPONSE
and its S-nitrosothiol derivatives play a role in inflammation. Cytokines such as interleukin-1 (IL-1) and
tumor necrosis factor promote the expression of the
inducible isoform of NOS and increase nitric oxide
production (10,19,22,28,29). Increased concentrations
of nitrites have recently been demonstrated in the
inflammatory joint fluids of patients with rheumatoid
arthritis (RA), indicating excessive local production of
NO at sites of chronic synovial proliferation (30).
Although in rodents, NO produced by macrophages
inhibits T cell proliferation (31-33), its effect on the
function of human lymphocytes is currently unknown.
In these studies we examined the effect of nitric
oxide and its stable derivative, S-nitrosoglutathione
(SNO-GSH), on the responses of activated human T
cells. The results indicate that these EDRFs may
inhibit T cell-mediated chronic inflammation such as
that in RA.
MATERIALS AND METHODS
Reagents. Nitric oxide solutions were prepared by
introducing NO gas into a tube (air-tight septum) containing
HEPES buffer (149 mM NaCl, 5 mM KOH, 10 mM HEPES,
1.2 mM MgCl,, 1.29 mM CaCl,), pH 7.4. The NO concentration was determined by Greiss reaction (34). S-nitrosoglutathione was prepared as described previously (35). Briefly,
red agarose resin suspended in an equal volume of Tris NaCl
(25 mM Tris, 0.9% NaCl), pH 8.1, was incubated with
different concentrations of glutathione (Sigma, St. Louis,
MO) for 5 minutes at 37°C. The reaction fluid was transferred
to a polypropylene Econo-column (Bio-Rad, Richmond,
CA), and eluate containing S-nitrosothiol was obtained.
SNO-GSH was measured after mercuric chloride diazotization, by Greiss reaction (34).
Culture media and media supplements were purchased from Gibco (Grand Island, NY), Ficoll-Hypaque
from Pharmacia LKB (Piscataway, NJ), and 3H-thymidine
from New England Nuclear (Boston, MA). Phytohemagglutinin (PHA), 3-isobutyl-1-methyl xanthine (IBMX), and flmonomethyl-L-arginine (L-NMA) were purchased from Calbiochem (La Jolla, CA). Human hemoglobin (Hgb),
methylene blue (MB), and L-leucine methyl ester (LME)
were purchased from Sigma, and the reagents for cyclic
GMP assay from Biomedical Technologies (Stoughton,
MA). Monoclonal anti-CD3 antibody (MAb) was purchased
from Coulter (Hialeah, FL). Reagents for determination of
IL-2 levels and IL-2 receptor expression were purchased
from Genzyme (Boston, MA).
Cell isolation and cell culture. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation. Human spleen T cells were isolated
from a normal spleen removed during an operative procedure. Spleen fragments were passed through a fine stainless
steel sieve and centrifuged to remove debris. Mononuclear
cells, from the single cell suspension, were isolated by
1415
Ficoll-Hypaque gradient centrifugation. T cells were isolated
by rosetting with neuraminidase-treated sheep red blood
cells, and aliquots of 5 x lo6 cells were frozen in liquid
nitrogen.
Monocytelmacrophaies were depleted by either a
2-hour incubation of 5 x lo6 PBMC over plastic (tissue
culture or microtiter plates) at 37°C or a 45-minute incubation in 5 mM LME at room temperature in serum-free
medium. The LME-treated cells were subsequently reconstituted with 5% macrophages to restore partial mitogen
responsiveness. Macrophages were isolated from PBMC
cultures either by adherence (as above), followed by repeated and thorough washing, or by Percoll gradient centrifugation.
Spleen T cells and PBMC were suspended at 1.2 x
lo6 cells in RPMI 1640 supplemented with 10% fetal calf
serum, penicillin (180 unitshl), streptomycin (100 pg/ml),
and L-glutamine (0.3 mg/ml), activated with PHA or with
CD3-specific MAb. Anti-CD3 antibody (10 pg/ml), in borate
buffered saline (0.1M borate, pH 8.4), was cross-linked to
the wells of flat-bottom microtiter plates (180 pVwe11) by
incubation at 37°C for 3 hours. The plates were washed 3
times with phosphate buffered saline and 170 pl of PBMC or
spleen T cell cultures (2 x 10’ cells/well) was added to the
coated wells.
For PHA activation, PBMC and spleen T cells were
treated with 2 pg/ml of purified PHA and cultured in microtiter plates (2 x 10’ cells/well). All cultures were incubated
at 37°C in an atmosphere containing 5% CO,.
T cell proliferation assay. T cell proliferation was
assayed by 3H-thymidine incorporation. Triplicate cultures
of anti-CD3-activated and PHA-activated cultures were
treated with various concentrations of SNO-GSH, glutathione, or nitric oxide 3 times a day for the first 48 hours of
culture, unless otherwise indicated. The cells were incubated at 37°C (in 5% CO,) for a total of 72 hours; 1 pCi/well
of ’H-thymidine was present for the last 18 hours of incubation. The cells were harvested onto glass-fiber filter paper,
and 3H-thymidine incorporation was determined by liquid
scintillation spectroscopy. Cell viability was determined
daily in all cultures by trypan blue exclusion.
Determination of IL-2 levels in culture supernatants.
The IL-2 content in culture supernatants was determined by
the enzyme-linked immunosorbent assay. Spleen T cells
were cultured as described above, treated with 12.5 or 25
p M SNO-GSH, and incubated at 37°C in 5% CO, for 6 , 24,
and 48 hours. After incubation, the plates were centrifuged
for 5 minutes at 2,500g and the IL-2 content of culture
supernatants (100 pl) was determined in duplicate, according
to the manufacturer’s instructions.
Determination of IL-2 receptor expression. IL-2 receptor expression on activated T cells was determined by
indirect immunofluorescence staining (36) and fluorescenceactivated cell sorter (FACS) analysis. T cells were activated
with PHA for 6,24, and 48 hours in the presence or absence
of 25 pM SNO-GSH. After each incubation period, IL-2
receptor expression was determined using a MAb against
IL-2 receptor (Genzyme), followed by incubation with
fluorescein-conjugated anti-mouse IgG and FACS analysis.
MERRYMAN ET AL
1416
Cyclic GMP accumulation in resting and activated
cells. SNO-GSH, reduced glutathione, and NO were added,
at various concentrations, to triplicate cultures of resting or
PHA-activatedcells ( 6 2 0 hours after stimulation) and incubated for 30 minutes at 37°C in 5% CO,, in the presence of
0.1 mM IBMX, unless otherwise indicated. Cyclic GMP was
assayed as described elsewhere (10). Briefly, cGMP was
extracted from the cells by rapid aspiration of the medium,
followed by addition of 200 pl of 0.1N HCl and incubation
for 30 minutes at 37°C. The extracts were frozen at -2O"C,
and for extraction of protein (before cGMP assay), 40 p1 of
1N NaOH was added to each well, and the wells were
incubated for 30 minutes at 37°C. The cGMP content of the
extracts was determined by radioimmunoassay after acetylation, using a rabbit antiserum and 12SI-labeledcGMP (obtained from Biomedical Technologies).
Statistical analysis. Statistical analysis was performed
by one-way analysis of variance with post hoc comparative
tests of selected pairs. The a value for hypothesis testing
was set at 0.05.
RESULTS
S-nitrosoglutathione inhibits DNA synthesis in
activated PBMC and spleen T cells. The effect of NO
and SNO-GSH on DNA synthesis of PHA-activated
PBMC and spleen T cells (E-rosette-positive) was
<15 seconds),
determined. Nitric oxide (half-life
S-nitrosoglutathione (TI,* >2 hours) (34), or reduced
glutathione was added 3 times daily (at 3-hour intervals) for the first 2 days of culture. Neither NO
(12.5-100 pM) nor GSH (12.5-25 pM) inhibited PHAdependent proliferation of PBMC or spleen T cells. In
contrast, SNO-GSH effectively inhibited proliferation
of both T cell populations.
As shown in Figure I, SNO-GSH, at 12.5 and
25 p M , inhibited DNA synthesis in PBMC (mean -+
SD 35 t 21% and 65 -+ 19% inhibition; P < 0.05 and P
< 0.001 versus baseline, respectively) and in spleen T
cells (50 t 11% and 75 -+ 15%; P < 0.001 and P <
0.001 versus baseline, respectively). Similar results
were obtained with CD3-specific MAb; 25 pM SNOGSH inhibited DNA synthesis in PBMC and T cell
cultures by 58% and 80%, respectively (n = 2). Significantly, hemoglobin, the nitric oxide inhibitor, reversed the inhibitory effect of SNO-GSH on DNA
synthesis (from 90% to 17% inhibition with Hgb) (n =
2). This effect is consistent with the hypothesis that
S-nitrosoglutathione acts via its reactive NO moiety.
Methylene blue, which inhibits guanyl cyclase, was
cytotoxic in the 3-day cultures (data not shown). As in
PHA-activated cultures, NO (25 pM) and GSH had no
effect on anti-CD3-induced T cell proliferation (data
not shown). SNO-GSH had no toxic effect on PBMC
m
2
X
I--I T
:I:
T
0
12.5
25
S - Nitrosoglutathione ( pM )
Figure 1. Effect of S-nitrosoglutathione (SNO-GSH) on DNA synthesis by phytohemagglutinin (PHA)-activated peripheral blood
mononuclear cells (PBMC) and spleen T cells. Cells (1.2 x lo6) were
activated with PHA (2 pg/ml) and cultured in 96-well flat-bottom
plates (200 pl/well) at 37°C in 5% CO, for 72 hours. SNO-GSH (12.5
and 25 pM) was added to triplicate cultures (3 times a day) during
the first 2 days of culture. DNA synthesis was determined by uptake
of 3H-thymidine(1 pCi/well), which was added 18 hours before the
end of culture. Values are the mean and SEM of 12-15 cultures (4 or
5 experiments). * = P < 0.001 versus baseline. Addition of the same
concentrations of glutathione and nitric oxide had no effect.
or spleen T cells, as determined by trypan blue exclusion as well as by the preserved capacity to produce
IL-2 (see below).
To determine whether the divergent effects of
NO and SNO-GSH on DNA synthesis were due to
their stability under the culture conditions, we measured the concentrations of both at l , 2 , 3 , and 4 hours
(26). Consistent with its short half-life in oxygenated
solution, 25 pM NO was not detected in culture
supernatants at subsequent time points. In contrast,
significant concentrations of 25 p M SNO-GSH were
found at all time points: K hour 17 p M , 2 hours 13.5
p M , 3 hours 9 p M , and 4 hours 6.6 p M . Thus, with our
experimental protocol whereby NO and SNO-GSH
are added to cultures 3 times daily to assess their
effects on DNA synthesis, these data indicate that only
SNO-GSH would be expected to be present at micromolar concentrations throughout the period of culture.
To examine the phase dependence of the Snitrosoglutathione effect, we varied the times of its
addition to culture. As shown in Figure 2, SNO-GSH
NO AND SNO-GSH MODULATE T CELL RESPONSE
120
T
60
40
20
Day 0
Day 1
Day2
Time of Addition of S - Nitrosoglutathione
( 50 pM 3x /day )
Figure 2. Phase-dependence of SNO-GSH-induced inhibition of
DNA synthesis. PHA-activated PBMC were cultured as described
in Figure I . SNO-GSH (50 tLM)was added to triplicate cultures 3
times a day on day 0, day 1, or day 2. DNA synthesis was
determined on day 3, following 18 hours of incubation with 'Hthymidine (1 @3/well). Values are the mean and SEM of 9-12
cultures (3 or 4 experiments). * = P < 0.05. See Figure 1 for
abbreviations.
had little effect on DNA synthesis when added during the first or second 20 hours of activation. However, significant inhibition (50%; P < 0.05) was obtained when SNO-GSH was added at 40-48 hours after
stimulation. These results suggest that the S-nitrosoglutathione-dependent inhibition of DNA synthesis
is activation-phase dependent, targeting late GI-S
phase events.
Effect of SNO-GSH on IL-2 production. IL-2
production and interaction with cell surface IL-2 receptors are essential steps in the induction of DNA
synthesis in activated T cells. We examined the effect
of S-nitrosoglutathione on IL-2 production and DNA
synthesis in duplicate sets of PHA-activated spleen T
cell cultures. Triplicate cultures in each set were
treated with 12.5 or 25 pM SNO-GSH for 6, 24, or 48
hours. At the end of each incubation period, culture
supernatants from one set were analyzed for IL-2
content, and the cells from the second set were used
for determination of DNA synthesis.
As expected, neither IL-2 nor DNA synthesis
was detectable in the 6-hour cultures (Figure 3). Twentyfour hours after PHA stimulation, however, increased
1417
and comparable levels of IL-2 were found in both
untreated (mean f SEM 267 -+ 197 pg/106 cells; P <
0.05 versus unstimulated control) and SNO-GSHtreated (249 +- 149 and 259 +- 99 pg/106 cells for 12.5
and 25 p M , respectively; P < 0.02 and P < 0.01 versus
controls, respectively) culture supernatants. At this
time point (24 hours), there was no significant increase
in DNA synthesis in either untreated or SNO-GSHtreated cultures. Following 48 hours of incubation,
significant DNA synthesis and IL-2 secretion were
observed in PHA-activated T cells (Figure 3). While
12.5 and 25 p M SNO-GSH inhibited DNA synthesis
by 75% and 95%, respectively, neither concentration
of SNO-GSH inhibited IL-2 synthesis (Figure 3).
These data indicate that the inhibition of DNA synthesis by SNO-GSH is independent of effects on IL-2
production.
SNO-GSH also did not affect IL-2 receptor
expression, as determined by FACS analysis of
treated and untreated cells stained with anti-IL-2
receptor antibody (Figure 4). Hemoglobin had no
significant effect on DNA synthesis, IL-2 production,
or IL-2 receptor expression (data not shown).
SNO-GSH inhibition of DNA synthesis is macrophage-independent. To determine whether the inhibitory effect of S-nitrosoglutathione was mediated
through the macrophage population, monocyte/
macrophages were depleted from PBMC cultures ei2500
IL-2
2000
'H-fhyrnidinr
upfake
I
-u
Y)
.0
1500
.
D
n.
N
1000
500
i
25
0
0
UNSTIMULATED
CONTROL
0
PHA STIMULATED
SNO-GSH
(pM)
Figure 3. Lack of effect of SNO-GSH on interleukin-2 (IL-2) production. PHA-activated spleen T cells were cultured as described in
Figure 1. SNO-GSH was added to replicate cultures for 6,24, and 48
hours, and IL-2 secretion and DNA synthesis were determined in
duplicate sets of cultures (see Materials and Methods). Values are
the mean and SEM of 9 determinations in 3 different experiments, at
48 hours. See Figure 1 for other definitions.
MERRYMAN ET AL
1418
3
2.5
*I
2.0
Nimc Oxide
1.5
FL Intensity
Figure 4. Lack of effect of SNO-GSH on interleukin-2 (IL-2) receptor expression. Anti-CD3 antibody-activated T cells were cultured
in 24-well plates as described in Figure 1. SNO-GSH (25 pkfl was
added to replicate cultures for 30 hours. PHA-activated celk (a),
PHA-activated and SNO-GSH-treated cells (b), and untreated,
resting control cells (c) were stained with anti-IL-2 receptor antibody by indirect immunofluorescence and analyzed by flow cytometry. FL = fluorescence; see Figure 1 for other definitions.
1.o
0.5
GSH
a
0.0
0
ther by adherence or by treatment with LME. As
expected, LME depletion of macrophages was nearly
complete; therefore, the LME-treated cells were reconstituted with macrophages at 5% density to restore
partial mitogen responsiveness (Table 1). Cultures
depleted by adherence contained 3-7% macrophages,
and thus also exhibited reduced responsiveness (DNA
synthesis) to PHA (Table 1). However, SNO-GSH
retained the capacity to inhibit proliferation of macrophage-depleted (by LME treatment or adherence)
PBMC. Indeed, macrophage depletion enhanced the
inhibition (Table 1) to a degree similar to that observed
for spleen T cells (Table 1 and Figure l), which are
also macrophage poor.
Experiments were performed to determine
whether macrophages were a source of endogenous
NO which modulated the T cell response, as has been
Table 1. Effect of macrophages on S-nitrosoglutathione (SNOGSH) inhibition of T cell proliferation*
Macrophage-depleted PBMC
Treatment
PBMC
Control
87,265
SNO-GSH 37,990
?
2
By adherence
By LME
13,740 19,022 5 1,841 38,116 2 3,725
14,585t 3,936 2 1,455t 11,942 2 5,108t
* Macrophages were depleted from peripheral blood mononuclear
cell (PBMC) cultures by adherence or by treatment with leucine
methyl ester (LME), as described in Materials and Methods. Both
undepleted and macrophage-depleted cell populations were activated with phytohemagglutinin and cultured in 96-well plates. SNOGSH (25 bhf)was added to triplicate cultures, and DNA synthesis
was determined. Values are the mean ? SD cpm x 103/cells.
t P < 0.001 versus controls.
?5
50
130
Concentration @M)
Figure 5. Effect of increasing concentrations of SNO-GSH, GSH,
and NO on the production of cGMP in PHA-activated PBMC.
Triplicate cultures were treated with the indicated concentrations of
SNO-GSH, GSH, or NO and incubated in the presence of 0.1 mM
3-isobutyl-i-methyl xanthine for 30 minutes at 37°C before cGMP
determination. Values shown are the mean
SEM of 9 cultures
from 3 experiments. Cyclic GMP values are elevated above control
(P< 0.01) with >25 pA4 NO and with >12.5 pA4 SNO-GSH. * = P
< 0.001 versus NO. See Figure I for other definitions.
*
described for rat “suppressor” macrophages (31-33).
The addition of L-NMA, an analog inhibitor of NO
synthesis, had no effect on the proliferation of PHAactivated PBMC; nor did reconstitution of L-NMAtreated macrophages with macrophage-depleted
PBMC (data not shown).
SNO-GSH and NO increase cGMP in PBMC and
spleen T cells. Nitric oxide activation of guanylate
cyclase in platelets and smooth muscle cells leads to
an increase in cellular cGMP levels (6,lO). It is through
this mechanism that NO exerts its biological effects in
a number of cell types (6,lO). We therefore performed
experiments to determine whether NO raised the
cGMP content of human T cells, as well as whether
SNO-GSH retained this NO property. Figure 5 shows
the effect of increasing concentrations of NO and
SNO-GSH on cGMP accumulation in PHA-activated
PBMC in the presence of XBMX, a phosphodiesterase
inhibitor. Both NO and SNO-GSH induced a significant increase in cGMP levels. SNO-GSH was a more
NO
1419
6
I
I
I
I
I
I
I
I
I
I
Spleen T Cells
PBMC
----
-t
O
L
I
1
I
I
c
I
I
I
I
Figure 6. Time course of SNO-GSH-induced and NO-induced cGMP accumulation in resting (0)
and PHAactivated ( 0 )PBMC and spleen T cells. SNO-GSH (25 CLM) was added to resting and activated cell cultures, in the
presence of 3-isobutyl-1-methyl xanthine (0.1 mM), incubated at 37°C in 5% CO,, and the cGMP content was
determined at the indicated times. Values are the mean f SEM of 8 replicate determinations from 2 experiments.
* = P < 0.001 versus control cultures. See Figure 1 for other definitions.
potent inducer of cGMP than was NO at all concentrations tested (P < 0.001). Furthermore, SNO-GSH
induced maximal cGMP accumulation at 12.5 pM (2.6
? 0.8 pmoles/106 cells; P < 0.001, versus controls),
while the maximal cGMP level for NO was obtained at
50-100 pikt (1.5 ? 0.4 pmoles/106 cells). GSH had no
appreciable effect (Figure 5).
Time course of cGMP accumulation. We next
studied the time course of S-nitrosoglutathioneinduced cGMP accumulation in resting and activated
PBMC and spleen T cells. The results are shown in
Figure 6. The SNO-GSH-induced increase in cGMP
levels was detectable in all cell populations within the
first 10 minutes of incubation. The cGMP increase in
resting and activated spleen T cells was similar, and
became optimal at 30 minutes (mean rt SEM 5.3 ? 1.4
and 5.6 k 1.3 pmoles/106 cells, respectively) (Figure
6). In contrast, the cGMP increase in both resting and
activated PBMC was maximal at 1 hour, and was
greater for the activated than the resting cells (5.3 ?
1.2 pmoles versus 3.8 t 1.0 pmoles; P < 0.05). The
maximal cGMP content of PBMC reached after 60
minutes was similar to that of spleen T cells after 30
minutes of incubation, which indicates a 30-minute lag
for cGMP production in PBMC (Figure 6).
The maximal cGMP levels remained unchanged
in all cultures for 3 hours and declined thereafter. The
rate of decline was nearly identical for resting and
activated cells in each cell population, and was greater
for PBMC than for spleen T cells. Cyclic GMP content
of both PBMC (0.93 ? 0.3 pmoles) and spleen T cells
(3.1 0.4 pmoles), however, remained significantly
above control values after 20 hours of incubation
(Figure 6). In contrast NO-induced cGMP accumulation in both resting and activated PBMC was maximal
in the first 10 minutes of incubation (1.0 t 0.2 and 1.2
? 0.5 pmoles, respectively; P < 0.01 and P < 0.05,
respectively, versus controls) and declined thereafter,
reaching control values in 1-2 hours.
NO inhibitors reverse SNO-GSH-induced cGMP
accumulation. To further confirm that S-nitrosoglutathione functioned as a nitric oxide surrogate, the
effect of NO inhibitors, methylene blue (a guanylate
cyclase inhibitor) and hemoglobin (a NO scavenger)
on cGMP accumulation was studied in activated
PBMC (Figure 7). Both inhibitors significantly reduced
SNO-GSH-induced cGMP accumulation. In the absence of inhibitors, cGMP increased to 4.1 2 0.35
pmoles, which is significantly greater than in untreated
controls (P < 0.001) (Figure 7). In the presence of 5 x
10-5M MB and 1 x 10-4M Hgb, the cGMP content
decreased to 0.466 +- 0.35 pmoles ( P < 0.001) and 0.76
*
MERRYMAN ET AL
1420
5
T
4
0
+
SNO - GSH ( 50 UM)
HEMOGLOBIN ( 1 ~ 1 0 ) 4 ~
METHYLEM BLUE ( 5xlO-’M )
-
+
+
+
+
Figure 7. Effect of inhibitors of soluble guanylate cyclase on SNOGSH-induced cGMP accumulation in PHA-activated PBMC. TO
triplicate cultures of PHA-activated (4hours) PBMC, 50 W S N O GSH was added, in the presence of 0.1 mM 3-isobutyl-1-methyl
xanthine. Guanylate cyclase inhibitors, hemoglobin and methylene
blue, were then added. Cultures were incubated for 30 minutes at
37°C in 5% CO, before cGMP determination. Values are the mean
and SEM of 9 cultures from 3 experiments. * = P < 0.001 versus
SNO-GSH-treated cells without inhibitors. See Figure 1 for other
definitions.
k 0.3 pmoles (P < O.OOl), respectively (Figure 7).
Under these experimental conditions, neither MB nor
Hgb was cytotoxic, based on trypan blue exclusion.
DISCUSSION
These studies demonstrate that S-nitrosoglutathione, the reaction product of nitric oxide and reduced
glutathione, an abundant sulfhydryl-containing tripeptide, inhibits the activation of human T cells and
functions as a nitric oxide surrogate. SNO-GSH inhibited T cell DNA synthesis both in PHA-activated and
CD3-specific MAb-activated PBMC and spleen T cell
cultures. Significantly, this inhibition was reversed by
the NO inhibitor, hemoglobin. SNO-GSH had no
effect on IL-2 synthesis or IL-2 receptor expression,
which suggests that its inhibition of DNA synthesis
may be activation-phase dependent (late G, early S
phase). This is supported by the observation that
SNO-GSH did not inhibit DNA synthesis when added
during the first 40 hours of incubation but was inhibitory when added at 40-48 hours after stimulation.
The SNO-GSH-dependent inhibition of DNA
synthesis appeared to be due to direct effects on T
cells. Depletion of macrophages from PBMC cultures
did not abrogate the inhibitory effect. In addition, the
extent of inhibition in macrophage-depleted cultures
was greater than that in undepleted PBMC and approached that in spleen T cells. This suggests that
macrophages may antagonize the effect of NO and its
S-nitrosothiol derivatives. Our recent observation that
oxidants produced by phagocytic cells degrade
S-nitrosothiols (34) is a possible mechanism which
may explain this observation. These features are currently under investigation.
NO added 3 times daily did not inhibit T cell
proliferation in these studies. It is likely that due to the
short half-life of NO, such “pulse” treatment was not
sufficient to exert inhibitory effects that might otherwise be observed if NO production were sustained. In
contrast to SNO-GSH, which was detectable at micromolar concentration at 4 hours following addition, NO
was undetectable in culture supernatants at all time
points studied. Interestingly, despite its rapid disappearance from culture supernatants, we demonstrated
increases in intracellular cGMP levels in response to a
single dose of NO. These increases were more modest
than those observed following the addition of SNOGSH and returned to baseline within 1 hour.
Although the pulse addition of NO could not be
shown to affect DNA synthesis, our data indicate that
S-nitrosoglutathione, which survived in cultures because of its half-life (>2 hours), exerted its effect
through NO-like mechanisms. First, the NO inhibitor,
hemoglobin, the iron-contaxning heme group of which
reacts directly with NO, reversed the SNO-GSHinduced inhibition of DNA synthesis. Second, like
NO, SNO-GSH raised intracellular cGMP levels. Indeed, it was more potent than NO both in terms of
peak cGMP levels achieved (2.6 t 0.8 pmoles at 12.5
pM SNO-GSH versus 1.5 ? 0.4 pmoles at 50 pM NO;
P < 0.001) and in the duration of cGMP elevation (1
hour versus 24 hours). Third, the NO inhibitors, Hgb
and MB blocked the capacity of SNO-GSH to raise
intracellular cGMP levels. The fact that cGMP levels
induced by SNO-GSH and NO differ markedly in both
concentration and duration suggests that the failure of
NO to inhibit T cell proliferation in our studies may be
directly related to its short half-life and its inability to
sustain elevated levels of ce:llular cGMP when administered at the intervals we used.
Further evidence that NO acts on human T cells
is provided by the recent report that the NOgenerating compounds, sodium nitroprusside and S-
NO AND SNO-GSH MODULATE T CELL RESPONSE
nitroso-N-acetyl penicillamine, inhibited DNA synthesis in activated PHMC in association with elevation of
cGMP levels (37). Interestingly, elevations of intracelMar cGMP are induced in platelets by S-nitrosothiols
and are associated with inhibition of platelet function
(8.9). These data, along with our previous observation
(26,341 that NO and SNO-GSH share the capacity to
inhibit human neutrophil superoxide anion production.
indicate that S-nitrosoglutathione may function as a
stable nitric oxide surrogate. Indeed, S-nitrosylation
by nitric oxide both of proteins and of low molecular
weight sulfhydryl-containing molecules, such as cysteine and glutathione, is increasingly recognized as a
mechanism by which the biological effects of NO are
sustained (4,8,9,38.39).
Although associated with elevations of cGMP,
our data do not indicate the mechanism by which
SNO-GSH inhibits T cell proliferation. The kinetics of
SNO-GSH inhibition of DNA synthesis and the absence of an SNO-GSH effect on IL-2 production or
IL-2 receptor expression suggest that SNO-GSH may
directly affect the enzymes involved in the process of
DNA replication. This is of interest, given the report
that the induction of a NO-generating pathway in
adenocarcinoma cells leads to inhibition of DNA synthesis and alteration of ribonucleotide reductase activity (29). Ribonucleotide reductase, a non-heme, ironcontaining protein, is a key enzyme for DNA synthesis
and consists of 2 subunits MI and M2. The expression
of the M2 subunit is cell cycle dependent (present at
high levels during S and G2 phases) and controls the
activity of the enzyme (40,41). Recently, it has been
shown that NO inhibits ribonucleotide reductase via a
direct reaction with the tyrosyl radical at the active
site of the enzyme, thus inactivating it (42). Our
experiments raise the analogous possibility that NO
inactivates a key enzyme, such as ribonucleotide
reductase, required for DNA synthesis in activated T
cells.
The possible role of macrophages in the SNOGSH-induced inhibition of T cell proliferation is of
interest. Although SNO-GSH inhibited DNA synthesis in both PBMC and spleen T cell cultures. the
degree of inhibition was significantly greater for macrophage-deficient spleen T cells than for PBMC. Moreover. while this inhibitory effect was highly consistent
for the spleen T cells, it varied significantly for PBMC
from different subjects, which suggests that macrophages may determine the stability of SKO-GSH and,
by extension, the stability of NO. The production of
NO by macrophages plays a central role in the regulation of rat T lymphocyte proliferation ( 3 1-33). How-
1421
ever, in this study, neither treatment of activated
PBMC with I,-NMA nor reconstitution of L-NMApretreated or untreated macrophages. with or without
lipopolysaccharide activation, had an effect on DNA
synthesis. whether in the presence or absence of GSH
and/or SNO-GSH or NO.
Thus, while our studies show that T cells are
capable of responding to NO, they do not show
definitively whether human macrophages are capable
of NO production sufficient to affect T cell responses.
This apparent failure of human macrophages to produce NO in our studies may be biologically significant.
indicative of differences between humans and rodents.
or it may simply reflect experimental conditions which
were insufficient to elicit NO production. Should human macrophages indeed be incapable of NO synthesis. alternative cellular sources of NO at sites of
inflammation would include neutrophils, endothelium.
fibroblasts. synoviocytes. and chondrocytes 15.15I7,28).
In summary. our data demonstrate that human
T cells respond to nitric oxide and its derivative
S-nitrosoglutathione. Significant inhibition of DNA
synthesis. in the absence of effects on IL-2 production
or IL-2 receptor expression, suggests that S-nitrosoglutathione exerts its effect during late G,-S phase
events. Prolonged elevations of intracellular cGMP
further suggest that 5-nitrosoglutathione. and possibly
other S-nitrosothiol derivatives of nitric oxide, are
stable sources of nitric oxide which inhibit T cell
responses at sites of chronic inflammation.
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