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SIN-1, a Nitric Oxide Donor, Ameliorates
Experimental Allergic Encephalomyelitis in
Lewis Rats in the Incipient Phase: The
Importance of the Time Window
Ling-Yun Xu, Jian-She Yang, Hans Link and Bao-Guo Xiao
J Immunol 2001; 166:5810-5816; ;
doi: 10.4049/jimmunol.166.9.5810
http://www.jimmunol.org/content/166/9/5810
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Copyright © 2001 by The American Association of
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References
SIN-1, a Nitric Oxide Donor, Ameliorates Experimental
Allergic Encephalomyelitis in Lewis Rats in the Incipient
Phase: The Importance of the Time Window1
Ling-Yun Xu, Jian-She Yang, Hans Link, and Bao-Guo Xiao2
N
itric oxide, when synthesized from L-arginine by the inducible NO synthase (iNOS),3 has been profoundly
studied in experimental allergic encephalomyelitis
(EAE), an animal model of human multiple sclerosis (MS). A cytotoxic role for NO in myelin destruction was first proposed. iNOS
gene expression and enzyme activity have been implicated in the
pathogenesis of MS and correlate with disease activity in EAE
(1–3). Inhibition of iNOS activity by the pharmacologic pathway
or intraventricular administration of antisense oligodeoxynucleotide resulted in suppression of EAE (4 –9).
On the other hand, some studies performed with MS patients
also showed that there was no correlation between raised serum
levels of nitrate/nitrite and magnetic resonance image activity, disease progression, or the development of brain atrophy (10). NO
was found to play an important part in the elimination of infiltrating inflammatory cells from lesions in the CNS in EAE (11) and
might be responsible for the spontaneous recovery from EAE in
Lewis rats (12, 13). Dendritic cell (DC)-derived NO was implicated in IL-4- and TGF-␤1-induced suppression of EAE (14, 15).
Moreover, administration of iNOS inhibitors caused some EAE-
Experimental Neurobiology and Neuroimmunology Units, Division of Neurology,
Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
Received for publication February 7, 2001. Accepted for publication February
20, 2001.
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.
resistant rodent strains to become highly susceptible to disease
induction (16), and EAE was exacerbated in mice lacking the
iNOS gene (17, 18). Most recent data indicated that oral treatment
of fully recovered EAE Lewis rats with N-methyl-L-arginine acetate, an iNOS inhibitor, leads to spontaneous relapse of EAE (12),
revealing that NO donor may be used to treat EAE and prevent
disease relapse.
Taken together, these studies indicate that NO may play multiple roles in EAE (for review, see Refs. 19 and 20). However, most
previous studies were performed in a negative pattern, i.e., either
pharmacologic inhibition or genetic inactivation of iNOS. To obtain further information about the role of NO in EAE, we now use
an NO donor, 3-morpholinosydnonimine (SIN-1), with the goal of
enhancing NO production during EAE development. We found
that when SIN-1 was given on days 5–7 postimmunization (p.i.),
i.e., during the incipient phase of EAE, clinical signs of EAE were
clearly reduced compared with those of PBS-treated control rats,
paralleled by reduction of macrophage and CD4⫹ T cell infiltrations within the CNS. Our data support the idea that NO plays a
critical role in the control of EAE. SIN-1 administration on days
5–7 p.i. enhanced NO production as well as IFN-␥ expression and
secretion by blood mononuclear cells (MNC). Simultaneously,
Ag- and mitogen-induced proliferation, and expression of MHC
class II, B7-1, and B7-2 were down-regulated. Augmented apoptosis among blood MNC was also observed in SIN-1-treated rats.
Materials and Methods
Reagents
1
This work was supported by grants from the Swedish Medical Research Council, the
Swedish MS Society, and Karolinska Institute Research Funds.
2
Address correspondence and reprint requests to Dr. Bao-Guo Xiao, Division of
Neurology, Karolinska Institute, Huddinge University Hospital, S141-86 Huddinge,
Stockholm, Sweden. E-mail address: [email protected]
3
Abbreviations used in this paper: iNOS, inducible NO synthase; DC, dendritic cells;
EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; SIN-1, 3-morpholinosydnonimine; MBP, myelin basic protein; MNC, mononuclear cells;
␻
L-NAME, N -nitrol-L-arginine methylester; p.i., postimmunization; SOD, superoxide
dismutase; O2⫺, superoxide; ONOO⫺, peroxynitrite; ELISPOT, enzyme-linked immunospot; AD, aminoguanidine.
Copyright © 2001 by The American Association of Immunologists
Guinea pig myelin basic protein (MBP) peptide covering aa residues
68 – 86 (MBP68 – 86; YGSLPQKSQRSQDENPV) was synthesized in an automatic Tecan/Syro synthesizer (Multisyntech, Bochum, Germany). SIN-1,
N␻-nitrol-L-arginine methylester (L-NAME), superoxide dismutase (SOD),
modified Griess reagent, Con A, and cytochrome c were purchased from
Sigma (St. Louis, MO), 2,6,8-trihydroxypurine (uric acid) was obtained
from KEBOLab (Stockholm, Sweden). Mouse anti-rat IFN-␥ mAb (DB1)
was purchased from Innogenetics (Ghent, Belgium). Anti-rat CD4 mAbs
were purified from culture supernatant of hybridoma clone W3/25. Anti-rat
macrophage mAbs (ED1) were purchased from Serotec (Oxford, U.K.).
0022-1767/01/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on
days 5–7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in
PBS-treated control rats and was accompanied by reduced ED1ⴙ macrophages and CD4ⴙ T cell infiltration within the CNS. Blood
mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-␥
production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were
down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In
vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and
peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates
disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on
NO synthase. The Journal of Immunology, 2001, 166: 5810 –5816.
The Journal of Immunology
Anti-nitrotyrosine mAb was obtained from Upstate Biotechnology (Lake
Placid, NY). PE-conjugated anti-rat MHC class II, FITC-conjugated antirat IFN-␥, PE-conjugated anti-mouse, and isotype control Abs were purchased from Serotec. Mouse-anti-rat B7-1 and B7-2 and PE-conjugated
anti-rat IL-4 mAb were obtained from PharMingen (San Diego, CA). Annexin V-FLUOS and propidium iodide were purchased from Roche
(Mannheim, Germany).
Animals
Lewis rats, 6 – 8 wk old, were purchased from Zentralinstitut fur Versuchstierzucht (Hannover, Germany).
Induction of EAE
Each rat was immunized s.c. at the tail root with 200 ␮l of inoculum
containing 25 ␮g of MBP68 – 86, 2 mg of Mycobacterium tuberculosis
(strain H37RA; Difco, Detroit, MI), 100 ␮l of saline, and 100 ␮l of IFA
(Difco). Rats were weighed and evaluated daily in a blinded fashion by at
least two investigators for the presence of clinical signs. Clinical scores of
EAE were graded according to the following criteria: 0, asymptomatic; 1,
flaccid tail; 2, loss of righting reflex with or without partial hind limb
paralysis; 3, complete hind limb paralysis; 4, moribund; and 5, dead.
Based on preliminary experiments of dosage, rats received i.p. injection of
SIN-1 only (0.1 mg/rat/day) or injection of SIN-1 plus uric acid (200 mg/
rat/day), SIN-1 plus SOD (5000 U/rat/day), or SIN-1 plus L-NAME (25
mg/rat/day) for 3 consecutive days from day 5 p.i. to day 7 p.i. Control rats
received i.p. injection of PBS (pH 7.4) only. At the same time, uric acid,
SOD, and L-NAME were administered at the same dosage and time points
to assess their effectiveness in inhibiting NO and scavenging superoxide
(O2⫺) and peroxynitrite (ONOO⫺).
Preparation of blood MNC
On day 14 p.i., peripheral blood was obtained from the tail vein. MNC
suspensions were prepared by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway). Cells were then washed three times
and resuspended in medium consisting of DMEM (Life Technologies,
Paisley, U.K.), supplemented with 1% MEM amino acids (Life Technologies), 2 mM glutamine (Flow, Irvine, U.K.), 50 IU of penicillin and 50
␮g/ml streptomycin (Life Technologies), and 10% (v/v) heat-inactivated
FCS (Life Technologies). Cells were then adjusted to 2 ⫻ 106/ml.
Measurement of nitrite
NO was assayed by measuring the end product, nitrite, which was determined by a colorimeter assay based on the Griess reaction. MNC (4 ⫻
105/200 ␮l) were incubated for 72 h in vitro with or without MBP68 – 86 at
37°C. Aliquots of cell culture supernatant (100 ␮l) were mixed with 100 ␮l
of Griess reagent at room temperature for 10 min. Absorbance was measured at 540 nm in an automated plate reader. The concentration of nitrite
was determined by reference to a standard curve of sodium nitrite (Sigma).
Samples incubated in the absence of cells were used as blanks. Assays were
performed in quadruplicate.
⫺
Measurement of O2 production
Production of O2⫺ was measured as the SOD-inhibitable reduction of cytochrome c (21, 22). MNC (2 ⫻ 106/ml) were incubated for 12 h at 37oC
in physiological salt solution (138 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.47 mM KH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 7.5 mM
glucose, pH 7.4) containing 200 ␮g of cytochrome c. After incubation,
supernatants were collected and centrifuged, and absorbance was measured
at 540 nm in an automated plate reader. Samples incubated in the absence
of cells were used as blanks. Assays were performed in quadruplicate.
Enumeration of MBP68 – 86-reactive IFN-␥-secreting cells by
enzyme-linked immunospot (ELISPOT)
ELISPOT assays were adopted for detection of IFN-␥ secretion at the
single-cell level. Nitrocellulose bottom microtiter plates (Millititer-HAM
plates; Millipore, Bedford, U.K.) were coated with 100-␮l aliquots of antirat IFN-␥ mAb (DB1) at 15 ␮g/ml. MNC suspensions (1 ⫻ 105 cells/200
␮l) were added to individual wells and incubated with or without
MBP68 – 86 (10 ␮g/ml). After 48 h of culture, the wells were extensively
washed. The plates were incubated with 100 ␮l of polyclonal rabbit anti-rat
IFN-␥ Ab (Innogenetics) diluted 1/500 for 4 h at room temperature. After
washing, the plates were incubated with biotinylated swine anti-rabbit IgG
(1/500; Dakopatts, Copenhagen, Denmark) and then with avidin-biotin per-
oxidase complex (1/200; Vector Laboratories, Burlingame, CA) followed
by peroxidase staining. The red-brown immunospots, which corresponded
to the cells that had secreted IFN-␥, were counted in a dissection
microscope.
Lymphocyte proliferation assays
Proliferative responses of MNC were examined by [3H]thymidine incorporation. Briefly, 200 ␮l of MNC suspensions (2 ⫻ 106/ml) were incubated
in 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) at
37°C in 5% CO2 with or without MBP68 – 86 (10 ␮g/ml) or Con A (5
␮g/ml). After 60 h, cells were pulsed with [3H]thymidine (1 ␮Ci/well;
Amersham, Little Chalfont, U.K.) for 12 h. Cells were harvested and
[3H]thymidine incorporation was measured in a liquid beta scintillation
counter.
Immunohistochemistry
On day 14 p.i., animals were sacrificed, and the spinal cords were dissected. Segments of lumbar spinal cord were snap-frozen in liquid nitrogen. Cryostat sections were cut at 10 ␮m and fixed in acetone for 10 min.
Endogenous peroxidase activity was inactivated with 0.03% H2O2 for 20
min. Nonspecific binding sites were further blocked with 1% blocking
reagent (Roche). The sections were incubated overnight in primary antiCD4 and ED1 Abs at a dilution of 1/100. Reactivity was detected with an
avidin-biotin peroxidase complex-reactive system (Vector Laboratories).
The specificity of the staining was tested by incubating sections without the
primary Abs. For each animal, three spinal cord sections were examined in
a blind fashion. Positive cells were counted by automatic video scanning
using Leica Q500 MC (Zeiss, Oberkochen, Germany).
To determine peroxynitrite formation, nitrotyrosine was detected by immunohistochemical techniques (23, 24). Anti-nitrotyrosine mAb was used
as primary Ab with 1/500 dilution overnight at 4oC. After incubation with
biotinylated anti-mouse IgG (1/200), the complex was visualized with an
avidin-biotin peroxidase complex-reactive system (Vector Laboratories).
Flow cytometry
For cell surface staining, 2 ⫻ 105 cells were incubated with PE-conjugated
anti-rat MHC II or unlabeled mouse anti-rat B7-1 or B7-2, followed by
PE-conjugated anti-mouse secondary Abs. All procedures were performed
in 1% BSA in PBS. For intracellular cytokine staining, 2 ⫻ 105 cells fixed
with 4% formaldehyde in phosphobuffer, permeabilized with 0.2% saponin
(Sigma), and then incubated with FITC-conjugated anti-rat IFN-␥ or PEconjugated anti-rat IL-4. All procedures were performed in 0.2% saponin/1% BSA in PBS. Ten thousand cells were analyzed by a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA).
Apoptosis assay
MNC (2 ⫻ 106/ml) were incubated with MBP68 – 86 (10 ␮g/ml) for 24 h,
and apoptosis was measured using annexin V-FLUOS (Roche) according
to the manufacturer’s instructions. Briefly, 2 ⫻ 105 cells were incubated in
100 ␮l of labeling solution containing 10 ␮l annexin-V-FLUOS and 10 ␮l
of propidium iodide for 15 min at room temperature. Cells were analyzed
on a FACScan.
Statistics
Differences between two groups were tested by Student’s t test. Differences
between more than two groups were tested by ANOVA. The level of significance was set at ␣ ⫽ 0.05.
Results
Suppression of clinical EAE by SIN-1
All PBS-treated rats immunized with MBP68 – 86 developed typical
clinical signs of acute EAE, with onset on days 9 –10 p.i. Acute
EAE peaked clinically on days 12–13 p.i. with a mean peak clinical score of 3.1. All rats totally recovered from clinical signs of
EAE by day 20 p.i., and no rats died.
In 12 rats receiving SIN-1 (0.1 mg/rat/day) on days 5–7 p.i., the
severity of clinical signs was reduced (mean peak clinical score,
1.4) compared with severity in PBS-treated 12 control EAE rats
( p ⬍ 0.05; Fig. 1). All rats developed signs of acute EAE, and
none of the rats died during the experiments. If the dosage of
SIN-1 was increased up to 1 mg/rat/day, no further clinical improvement was observed (mean peak clinical score, 1.4; p ⬍ 0.05
compared with severity in PBS-treated control EAE rats; p ⬎ 0.05
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Administration of SIN-1
5811
5812
NO DONOR IN EAE
compared with that of 0.1 mg/rat/day SIN-1). If the dosage was
decreased to 0.01 mg/rat/day, no difference in severity of clinical
signs was observed between these rats and control EAE rats receiving PBS (mean peak clinical score, 2.5, p ⬎ 0.05; Fig. 1a). In
preliminary experiments SIN-1 was also given on days 0 –2 p.i. at
the same dosage without any difference in clinical severity compared with that in PBS-treated control EAE rats (data not shown).
In vitro, SIN-1 is known to simultaneously produce both NO
and O2⫺, which combine to form ONOO⫺. Therefore, the question
arises of whether SIN-1 exerts its effect via NO or other products.
We added uric acid (the scavenger of ONOO⫺, 200 mg/rat/day)
and SOD (the scavenger of O2⫺, 5000 U/rat/day) to investigate the
pathway of SIN-1 function. We also added L-NAME (25 mg/rat/
day) to examine whether NOS is involved in SIN-1 function. As
shown in Fig. 1b, addition of these scavengers did not alter the
suppressive effect of SIN-1 on EAE (the difference in mean peak
clinical score among SIN-1, SIN-1 plus uric acid, SIN-1 plus SOD,
and SIN-1 plus NAME is not statistically significant; however, the
mean peak clinical scores of these four groups are all significantly
lower than that of the PBS-treated group, with p ⬍ 0.01, respectively), demonstrating that the function of SIN-1 was due to NO,
not to other intermediates, and is NOS independent.
SIN-1 administration enhanced NO production
As shown in Fig. 2, higher levels of nitrite were detected in cell
supernatants of SIN-1-treated animals both spontaneously and
upon stimulation with MBP68 – 86 ( p ⬍ 0.001). Thus, SIN-1 administration enhanced NO production in vivo. NO can be trans-
FIGURE 2. Production of NO (a), O2⫺ (b), and ONOO⫺ (c) after administration of NAME, SOD, and uric acid during EAE in Lewis rats. For
3 consecutive days between days 5 and 7 p.i., rats received i.p. injection of
SIN-1 (0.1 mg/rat/day), uric acid (200 mg/rat/day), SOD (5000 U/rat/day),
or L-NAME (25 mg/rat/day). The control EAE rats received i.p. injection
of only PBS between days 5 and 7 p.i. On day 14 p.i., blood MNC were
prepared from four rats of different groups. The measurements of NO, O2⫺,
and ONOO⫺ are described in Materials and Methods. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01; ⴱⴱⴱ, p ⬍ 0.001.
formed by reacting with another enzymatically produced free radical, O2⫺, to form ONOO⫺, which has been implicated in EAE.
Fig. 2 shows that administration of SIN-1 did not induce O2⫺ and
ONOO⫺ formation, although it can increase NO production, indicating that SIN-1 is likely to behave as an NO donor in vivo.
NAME inhibited the production of NO ( p ⬍ 0.001), while SOD
and uric acid reduced the formation of O2⫺ and ONOO⫺ in vivo
( p ⬍ 0.01 and p ⬍ 0.05, respectively), suggesting the effects of
these molecules to inhibit NO production and scavenge ONOO⫺
and O2⫺ formation. However, NAME (25 mg/rat/day), uric acid
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FIGURE 1. Effects of administration of SIN-1 on acute EAE in Lewis
rats. For 3 consecutive days between days 5 and 7 p.i., rats received i.p.
injection of a) SIN-1 (0.01, 0.1, or 1 mg/rat/day), b) SIN-1 (0.1 mg/rat/day)
plus uric acid (200 mg/rat/day) or SOD (5000 U/rat/day) or L-NAME (25
mg/rat/day). The control EAE rats received i.p. injection of only PBS between days 5 and 7 p.i. Results are from three independent experiments
with identical results (n ⫽ 12; four rats per group per experiment).
The Journal of Immunology
5813
(200 mg/rat/day), and SOD (5000 U/rat/day) did not influence clinical signs of EAE (data not shown). These results demonstrate that
SOD and uric acid can scavenge ONOO⫺ and O2⫺ formation, but
did not influence the clinical severity of EAE.
SIN-1 decreased the infiltration of inflammatory cells within
the CNS
Inflammatory cells are found within the CNS in acute EAE and are
associated with the clinical signs. We examined the infiltrations of
ED1⫹ macrophages and CD4⫹ T cells in spinal cord sections from
rats receiving SIN-1 and PBS. The infiltrations of ED1⫹ and
CD4⫹ T cells in spinal cord sections from SIN-1-treated rats
(mean, 44 ⫾ 12 and 35 ⫾ 8 cells/cm2, respectively) were clearly
reduced compared with those in PBS-treated control EAE rats
(mean, 106 ⫾ 22 and 68 ⫾ 14 cells/cm2, respectively; p ⬍ 0.05;
Fig. 3). Thus, SIN-1 administration inhibited the clinical severity
of EAE and reduced the infiltration of inflammatory cells within
the CNS.
SIN-1 administration induced MBP68 – 86-reactive IFN-␥
expression and secretion
We evaluated intracellular IFN-␥ and IL-4 expression of blood
MNC after in vitro incubation with MBP68 – 86 for 24 h. As shown
in Fig. 4a, the percentage of intracellular IFN-␥⫹ cells from SIN1-treated rats was almost twice that of cells from PBS-treated control EAE rats (15.18 vs 8.35%). No difference was found in the
percentage of intracellular IL-4⫹ cells between the two groups
(1.85 vs 1.23%).
As shown in Fig. 4b, ELISPOT assays revealed that numbers of
MBP68 – 86-reactive IFN-␥-secreting blood MNC were higher in
SIN-1-treated rats than in PBS-treated control EAE rats (28 ⫾ 3 vs
13 ⫾ 2%; p ⬍ 0.05). IL-4-secreting cells were undetectable by
ELISPOT assays in both SIN-1- and PBS-treated rats.
SIN-1 inhibited proliferative responses
Blood MNC were separated as described above, and proliferative
responses were measured. Upon stimulation with MBP68 – 86 or the
T cell mitogen Con A, proliferative responses were reduced in
SIN-1-treated rats compared with those in PBS-treated rats (Fig. 5;
p ⬍ 0.05), although the difference may not be biologically significant. SIN-1 administration thus down-regulated Ag- and mitogeninduced cell proliferation.
FIGURE 4. Cytokine production. On day 14 p.i. (mean clinical score:
PBS-treated control EAE rats, 3.5; SIN-1-treated rats, 1.3; p ⬍ 0.05), blood
MNC were prepared from four rats that had received PBS or SIN-1 on days
5–7 p.i. A, Intracellular IFN-␥ and IL-4 expressions were examined using
FACScan; B, IFN-␥ secretion was visualized using ELISPOT assay. Results (mean ⫾ SD) are representative of three independent experiments
with identical results. ⴱ, p ⬍ 0.05.
SIN-1 administration down-regulated surface expression of
MHC class II, B7-1, and B7-2
Blood MNC was separated on day 14 p.i. from rats that received
SIN-1 and PBS injections between days 5 and 7 p.i. FACScan
showed that percentages of MHC class II⫹, B7-1⫹, and B7-2⫹
cells in SIN-1-treated rats (2.85, 0.71, and 1.10%, respectively)
were lower than those in PBS-treated control EAE rats (14.76,
6.32, and 5.17%, respectively; Fig. 6). Thus, SIN-1 administration
between days 5 and 7 p.i. down-regulated the expression of surface
molecules associated with Ag presentation and T cell activation.
SIN-1 augmented apoptosis
On day 14 p.i., blood MNC were separated from SIN-1- and PBStreated rats. Upon stimulation with MBP68 – 86, cells were incubated in vitro for 24 h. Percentages of apoptotic cells were measured by annexin-V-FLUOS staining. As shown in Fig. 7, the
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FIGURE 3. Infiltration of inflammatory cells within the CNS. On day
14 p.i. (mean clinical score: PBS-treated control EAE rats, 3.5; SIN-1treated rats, 1.3; p ⬍ 0.05), four rats from different groups were sacrificed,
and lumbar spinal cords were dissected. Infiltrating ED1⫹ macrophages
and CD4⫹ T cells were detected by immunohistochemical staining. Results
(mean ⫾ SD) are representative of three independent experiments with
identical results. ⴱ, p ⬍ 0.05.
5814
percentage of apoptotic cells among MNC from SIN-1-treated rats
was 3.66 ⫾ 0.64% and that of apoptotic cells from PBS-treated
rats was 1.57 ⫾ 0.23 ( p ⬍ 0.01). Therefore, SIN-1 administration
can enhance apoptosis among MBP68 – 86-reactive blood MNC.
Discussion
In vitro, SIN-1 slowly decomposes to release both NO and O2⫺,
thereby producing ONOO⫺. In vivo, various antioxidant substances exist, including vitamin E, vitamin C, and reduced gluta-
FIGURE 6. Surface molecule expression. On day 14 p.i. (mean clinical
score: PBS-treated control EAE rats, 3.5; SIN-1-treated rats, 1.3; p ⬍
0.05), blood MNC were prepared from four rats that had received PBS or
SIN-1 on days 5–7 p.i. Cells were incubated in vitro with MBP68 – 86 for
24 h. The percentages of MHC II⫹, B7-1⫹, and B7-2⫹ cells were measured
using FACScan. Results are representative of four samples with identical
results.
FIGURE 7. Apoptosis evaluation. On day 14 p.i. (mean clinical score:
PBS-treated control EAE rats, 3.5; SIN-1-treated rats, 1.3; p ⬍ 0.05), blood
MNC were prepared from four rats that had received PBS or SIN-1 on days
5–7 p.i. Cells were incubated in vitro with or without MBP68 – 86 for 24 h.
Apoptosis was examined using annexin V-FLUOS staining. Results
(mean ⫾ SD) are representative of three independent experiments with
identical results. ⴱⴱ, p ⬍ 0.01.
thione (GSH) (25). Peroxynitrite formation by SIN-1 can be inhibited, and SIN-1 induces the release of NO in the presence of
GSH (26). Therefore, SIN-1-generated peroxynitrite is scavenged
by antioxidants and might not exhibit sufficient physiologic and
pathologic effects. Singh et al. (27) demonstrated that SIN-1 is
converted from a peroxynitrite donor, in aerobic solutions, to an
NO donor in vivo. Our results also demonstrate that SIN-1 only
induced NO production, but did not increase O2⫺ and ONOO⫺
formation. By which mechanisms does SIN-1 behave more like an
NO donor than a peroxynitrite donor in vivo? SIN-1 may react
with heme proteins and other electron acceptors in biological systems to produce NO without the concomitant production of O2⫺.
Oxidized heme proteins can accept an electron from SIN-1 and
stimulate NO release. Thus, at the relatively low oxygen concentrations in vivo, SIN-1 is likely to behave as an NO donor.
In addition, ONOO⫺ has a short half-life (28). ONOO⫺ formed
from SIN-1 reacts with hydroxyl-substituted molecules such as
glucose or glycerol to form mono- and dinitrite esters (29, 30).
HEPES-containing buffer was also reported to stimulate NO formation from peroxynitrite in a reaction catalyzed by cupric ions
(31). The peroxynitrite also stimulates guanylate cyclase and induces the production of cGMP, which, in turn, induces the regeneration of NO (26).
To further define whether NO is responsible for the inhibition of
EAE by SIN-1, the superoxide scavenger SOD and the peroxynitrite scavenger uric acid were added to SIN-1 solution to investigate the effect of SIN-1. Addition of these scavengers of superoxide and peroxynitrite did not alter the suppressive effect of SIN-1
on EAE, demonstrating that the suppressive function of SIN-1 was
due to NO and not to other intermediates. Administration of
NAME, uric acid, or SOD can inhibit NO production and O2⫺ and
ONOO⫺ formation in vivo, but did not influence clinical signs of
acute EAE in Lewis rats (data not shown). We also used L-NAME
to examine whether NOS is involved in SIN-1 function. Our results indicate that SIN-1-released NO in vivo is NOS independent.
Taken together, these findings indicate that SIN-1 suppressed the
development of incipient EAE mainly through SIN-1-derived NO,
without NOS dependence.
In a previous study by Okuda et al. (32), aminoguanidine (AD),
a selective iNOS inhibitor, was given to mice with actively induced EAE. Administration of AD by i.p. or intracisternal injection from days 2 to 12 p.i. produced a significant delay in the onset
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Proliferative response. On day 14 p.i. (mean clinical score:
PBS-treated control EAE rats, 3.5; SIN-1-treated rats, 1.3; p ⬍ 0.05), blood
MNC were prepared from four rats that had received PBS or SIN-1 on days
5–7 p.i. Proliferative responses were evaluated by measuring [3H]thymidine incorporation upon stimulation with MBP68 – 86 (10 ␮g/ml) or Con A
(5 ␮g/ml). Results (mean ⫾ SD) are representative of three independent
experiments with identical results. ⴱ, p ⬍ 0.05.
NO DONOR IN EAE
The Journal of Immunology
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of EAE. On the other hand, administration of AD by i.p. or intracisternal injection for 10 days after the onset of clinical EAE enhanced the clinical severity and mortality rate and hastened the
onset of relapse. These data suggested that NO plays different roles
during the induction and progression phase of EAE. In preliminary
studies we delivered SIN-1 from days 0 to 2 p.i. without observing
any influence on the course of EAE. However, when SIN-1 was
given on days 5–7 p.i., i.e., during the incipient phase, both clinical
and histologic signs of EAE were reduced compared with those in
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NO may selectively inhibit the development of primed Th1 cells
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SIN-1 administration resulted in enhanced production of NO as
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Since the rate of cell proliferation actually depends on the balance between cell death vs cell division and cell cycle progression,
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The role of NO in the immune system comprises both regulatory
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be of importance in developing new therapeutic strategies.
5815
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NO DONOR IN EAE