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The Journal of Immunology, 2001, 166: 5810-5816.
Copyright © 2001 by The American Association of Immunologists

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

Experimental Neurobiology and Neuroimmunology Units, Division of Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ED1+ macrophages and CD4+ T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-{gamma} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric 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, 2, 3). Inhibition of iNOS activity by the pharmacologic pathway or intraventricular administration of antisense oligodeoxynucleotide resulted in suppression of EAE (4, 5, 6, 7, 8, 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-{beta}1-induced suppression of EAE (14, 15). Moreover, administration of iNOS inhibitors caused some EAE-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-{gamma} 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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{omega}-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-{gamma} 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.). Anti-nitrotyrosine mAb was obtained from Upstate Biotechnology (Lake Placid, NY). PE-conjugated anti-rat MHC class II, FITC-conjugated anti-rat IFN-{gamma}, 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 µl of inoculum containing 25 µg of MBP68–86, 2 mg of Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI), 100 µl of saline, and 100 µl 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.

Administration of SIN-1

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 µg/ml streptomycin (Life Technologies), and 10% (v/v) heat-inactivated FCS (Life Technologies). Cells were then adjusted to 2 x 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 x 105/200 µl) were incubated for 72 h in vitro with or without MBP68–86 at 37°C. Aliquots of cell culture supernatant (100 µl) were mixed with 100 µl 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 x 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 µg 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-{gamma}-secreting cells by enzyme-linked immunospot (ELISPOT)

ELISPOT assays were adopted for detection of IFN-{gamma} secretion at the single-cell level. Nitrocellulose bottom microtiter plates (Millititer-HAM plates; Millipore, Bedford, U.K.) were coated with 100-µl aliquots of anti-rat IFN-{gamma} mAb (DB1) at 15 µg/ml. MNC suspensions (1 x 105 cells/200 µl) were added to individual wells and incubated with or without MBP68–86 (10 µg/ml). After 48 h of culture, the wells were extensively washed. The plates were incubated with 100 µl of polyclonal rabbit anti-rat IFN-{gamma} 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 peroxidase complex (1/200; Vector Laboratories, Burlingame, CA) followed by peroxidase staining. The red-brown immunospots, which corresponded to the cells that had secreted IFN-{gamma}, were counted in a dissection microscope.

Lymphocyte proliferation assays

Proliferative responses of MNC were examined by [3H]thymidine incorporation. Briefly, 200 µl of MNC suspensions (2 x 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 µg/ml) or Con A (5 µg/ml). After 60 h, cells were pulsed with [3H]thymidine (1 µCi/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 µm 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 anti-CD4 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 x 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 x 105 cells fixed with 4% formaldehyde in phosphobuffer, permeabilized with 0.2% saponin (Sigma), and then incubated with FITC-conjugated anti-rat IFN-{gamma} or PE-conjugated 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 x 106/ml) were incubated with MBP68–86 (10 µg/ml) for 24 h, and apoptosis was measured using annexin V-FLUOS (Roche) according to the manufacturer’s instructions. Briefly, 2 x 105 cells were incubated in 100 µl of labeling solution containing 10 µl annexin-V-FLUOS and 10 µl 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 {alpha} = 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go). 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 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. 1Goa). 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).



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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).

 
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. 1Gob, 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. 2Go, 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 transformed by reacting with another enzymatically produced free radical, O2-, to form ONOO-, which has been implicated in EAE. Fig. 2Go 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 (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.



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  		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. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
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. 3Go). Thus, SIN-1 administration inhibited the clinical severity of EAE and reduced the infiltration of inflammatory cells within the CNS.



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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-1-treated 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. *, p < 0.05.

 
SIN-1 administration induced MBP68–86-reactive IFN-{gamma} expression and secretion

We evaluated intracellular IFN-{gamma} and IL-4 expression of blood MNC after in vitro incubation with MBP68–86 for 24 h. As shown in Fig. 4Goa, the percentage of intracellular IFN-{gamma}+ cells from SIN-1-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%).



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  		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-{gamma} and IL-4 expressions were examined using FACScan; B, IFN-{gamma} secretion was visualized using ELISPOT assay. Results (mean ± SD) are representative of three independent experiments with identical results. *, p < 0.05.

 
As shown in Fig. 4Gob, ELISPOT assays revealed that numbers of MBP68–86-reactive IFN-{gamma}-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. 5Go; p < 0.05), although the difference may not be biologically significant. SIN-1 administration thus down-regulated Ag- and mitogen-induced cell proliferation.



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  		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 µg/ml) or Con A (5 µg/ml). Results (mean ± SD) are representative of three independent experiments with identical results. *, 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. 6Go). 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.



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  		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.

 
SIN-1 augmented apoptosis

On day 14 p.i., blood MNC were separated from SIN-1- and PBS-treated 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. 7Go, the 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.



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  		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. **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 glutathione (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 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 PBS-treated control rats, further suggesting that SIN-1 has different effects on different stages of EAE, thereby reflecting an important time window. Our results might explain the concept that NO may selectively inhibit the development of primed Th1 cells and prevent the amplification of such cells (33).

SIN-1 administration resulted in enhanced production of NO as well as IFN-{gamma} by blood MNC. IFN-{gamma} is a well-known NO inducer. In vitro, IFN-{gamma} promoted NO production by DC, macrophages, and neutrophils (34, 35, 36, 37, 38). A most recent study in IFN-{gamma}R-/- mice showed that IFN-{gamma} down-regulates EAE by inducing iNOS and subsequent NO production both by macrophages in the periphery and by microglia and astrocytes in the target tissue (39). NO has the capacity to suppress IFN-{gamma} production (40). However, in vivo treatment with L-arginine enhanced IFN-{gamma} mRNA expression, which was detected earlier in EAE rats than in control rats (41). Therefore, the detected levels of NO and IFN-{gamma} production might be a comprehensive effect of mutual interactions between them.

Proliferation of blood MNC was reduced after SIN-1 administration. This is consistent with the widely accepted concept that excessive NO production inhibits cell proliferation. Elevated NO levels were responsible for suppressed T cell proliferation in Listeria monocytogenes infection (42) and in tumor-induced immunosuppression (43). Inhibition of iNOS activity restores lymphocyte proliferative responses (44). In vitro addition of the NO donor S-nitrosyl penicillamine suppressed the proliferation of Th1 clone cells (45). Sicher et al. (46) reported that NO modulated Ag presentation by down-regulating the expression of MHC class II molecules on APCs. In this study MHC class II, B7-1, and B7-2 molecules were down-regulated after SIN-1 administration between days 5 and 7 p.i. These molecules are crucial for effective Ag presentation and T cell activation. Thus, NO-induced suppression of these molecules might contribute to reduced cell proliferation and T cell activation.

Since the rate of cell proliferation actually depends on the balance between cell death vs cell division and cell cycle progression, it is not surprising that NO has been shown to act as an important inducer of programmed cell death. Thymic DC use nitrergic mechanisms for T cell deletion (47). In vitro studies demonstrated that high levels of NO induced apoptosis in murine peritoneal macrophages, splenic T cells, as well as thymocytes (48, 49, 50). Both in vivo and in vitro studies indicated that DC induced autoreactive T cell apoptosis through the NO pathway (13, 14, 15).

The role of NO in the immune system comprises both regulatory and effector functions. The regulatory functions include immunosuppressive effects (inhibition of lymphocyte proliferation), while effector functions include immunopathologic effects (tissue destruction) and immunoprotective activities (apoptosis of autoreactive T cells). Our study of the effects of NO donor on Lewis rat EAE indicates that NO may play an important role in down-regulating EAE. However, the role of NO in EAE is not the same in different phases of the disease, but changes according to the immunological status. This might also be true in human MS and may be of importance in developing new therapeutic strategies.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council, the Swedish MS Society, and Karolinska Institute Research Funds. Back

2 Address correspondence and reprint requests to Dr. Bao-Guo Xiao, Division of Neurology, Karolinska Institute, Huddinge University Hospital, S141-86 Huddinge, Stockholm, Sweden. Back

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{omega}-nitrol-L-arginine methylester; p.i., postimmunization; SOD, superoxide dismutase; O2-, superoxide; ONOO-, peroxynitrite; ELISPOT, enzyme-linked immunospot; AD, aminoguanidine. Back

Received for publication February 7, 2001. Accepted for publication February 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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