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,
,
*
Neurosciences Research Unit, Canberra Hospital, Woden, Australia;
John Curtin School of Medical Research, Australian National University, University of Sydney Canberra Clinical School, Canberra, Australia; and
Praxis Pharmaceuticals, Canberra, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EAE is an autoimmune CD4+ T cell-mediated disease of the CNS that is often used as a model of the human demyelinating disease multiple sclerosis (10). Pathologically, EAE is characterized by infiltration of the CNS by lymphocytic and mononuclear cells, a breakdown in blood-brain barrier permeability, astrocytic hypertrophy (11), and demyelination (12), which cumulatively contribute to clinical expression of disease. Clinically, the disease often manifests as an ascending paralytic disease of the hindlimbs with occasional forelimb involvement. The clinical course of the disease varies depending on the animal species and strain and type of Ag used for immunization (13).
Myelin basic protein in CFA (MBP-CFA)-induced EAE in the Lewis rat is an acute monophasic disease from which affected animals fully recover. This recovery is associated with an absence of spontaneous relapses and a long-term resistance to active reinduction of disease (14, 15, 16). Previously, we have reported (17, 18) increased serum levels of reactive nitrogen intermediates (RNIs), indicators of increased NO production, during the recovery phase of MBP-CFA-induced EAE in the Lewis rat. These levels remained elevated after the recovery period and increased even further early after a rechallenge with MBP-CFA. All rechallenged animals were totally refractory to a second episode of disease (18). Oral treatment of rats with a NOS inhibitor, N-methyl-L-arginine acetate (L-NMA), beginning at peak disease on day 11 postprimary immunization, resulted in significant prolongation of disease and an alteration in the presentation of clinical symptoms from that of solely hindlimb paresis/paralysis to severe forelimb involvement as well. It was found that these animals recovered only after the cessation of L-NMA treatment and the consequent rise in systemic NO production. Treatment of fully recovered rats with L-NMA beginning 24 h before a rechallenge with MBP-CFA led to decreased serum RNI levels and resulted in a second episode of EAE in 100% of otherwise totally resistant animals. Intriguingly, L-NMA treatment of fully recovered rats, even in the absence of a rechallenge immunization, led to a second clinical episode of disease.
Further studies on this model have now shown that animals recovered from actively induced EAE and treated with L-NMA, whether or not they are reimmunized with MBP-CFA, develop not only a second episode of disease, but this disease is a chronic relapsing one observed over many months. Unlike these animals, however, rats recovered from passively induced disease do not relapse following treatment, suggesting the need for a peripheral Ag depot to induce a second episode of disease. The mechanism of reinduction of disease by NOS inhibitors has been examined, and evidence suggests that a T lymphocyte escape from proliferation inhibition by NO may play a role.
We also demonstrate in this study that clinical signs of EAE in L-NMA-treated recovered rats do not appear during treatment with the inhibitor, but only after treatment is stopped. While receiving L-NMA, rats that show no clinical signs of disease nevertheless have quantitatively and qualitatively the same CNS inflammatory infiltrates as rats whose treatment was terminated 3 days earlier and who have severe clinical signs. The former animals also have a similar proportion of iNOS-producing cells in the CNS as do the latter, but significantly less RNIs in their cerebrospinal fluid (CSF). These findings suggest a dual role for NO in regulation of pathology in EAE that is dependent on site and timing of production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lewis rats (8–12 wk old) were obtained from the Animal Breeding Establishment at the Australian National University. They were bred under pathogen-free conditions and subsequently maintained in either the Animal Holding Facility at The Canberra Hospital or at the John Curtin School of Medical Research. Throughout the experiment, food and water were provided ad libitum, and they were housed under 12 hourly light and dark cycles.
Induction of active EAE
MBP was purified from frozen guinea pig spinal cord, according to the method of Eylar et al. (19). Guinea pig MBP in saline was emulsified in an equal volume of incomplete Freund’s adjuvant containing 4 mg/ml heat-killed Mycobacterium butyricum. Rats were anesthetized before immunization with 100 µl of emulsion into each hind footpad for the initial induction of EAE. For rechallenge, rats were immunized with 50 µl of emulsion into each front footpad and 100 µl intradermally in the nuchal region. Total dose received for each immunization per rat was 25 µg of guinea pig MBP and 400 µg of M. butyricum. Both routes of immunization produce clinical EAE of equal severity inclusive with the day of onset, incidence, and development of resistance.
Passive EAE
Donor rats were immunized with MBP-CFA, and spleens were removed day 10 postimmunization. Single-cell suspensions were prepared and cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% heat-inactivated FCS, 2.5 x 10-5 2-ME, 1 µg/ml penicillin-streptomycin-neomycin solution, and 2 µg/ml Con A at 37°C, 5% CO2 for 72 h. Cells were collected into HBSS and washed twice before counting and injecting the resuspended cells at a concentration of 40 x 106 viable cells in 500 µl of HBSS.
Evaluation of clinical signs of EAE
Rats were examined on a daily basis, and clinical scores were recorded from day 7 postimmunization to day 22. Scores were then recorded every other day until rechallenge at day 35, in which scoring was resumed on a daily basis. Clinical disease severity was assessed and scored as previously described (15) using a scale from 1 to 5 as follows: 0, asymptomatic; 1, flaccid distal half of tail; 2, entire tail flaccid; 3, ataxia, difficulty in righting; 4, hindlimb weakness; 5, hindlimb paralysis. Where necessary, the score was divided to indicate the disease score falling between two categories, e.g., 3.5.
Inhibition of NO production with L-NMA
L-NMA was synthesized using the method outlined by Patthy et al. and described in Ref. 17 . Lewis rats were housed individually and given L-NMA via their drinking water, as outlined in Results. The concentration of L-NMA needed to reduce RNI levels in MBP-CFA-immunized animals to that of naive animals has previously been established as 15 mM/day with the volume of fluid consumed between 15 and 25 ml/rat (14). As the initial anesthesia and immunization procedure causes the animals to temporarily reduce their fluid consumption by half, it is necessary to double the concentration of L-NMA in the drinking water for 24 h following immunization. The L-NMA solution was prepared daily and filter sterilized, and 25 ml was decanted into sterile 50-ml tubes. The daily volume consumed per rat was recorded at the same time each day and expressed as milligrams of L-NMA per rat per day.
Measurement of NO production
The level of nitrate and nitrite in plasma samples was determined as an indirect measurement of NO production in vivo, as outlined by Rockett et al. (20), and modified and described in detail by Cowden et al. (17). Briefly, 30-µl aliquots of serum were added in duplicate to a V-bottom microplate (Nunc, Roskilde, Denmark). Standard curves were generated using normal dialyzed rat plasma to which sodium nitrite or sodium nitrate had been added at concentrations of 1 mM to 1 µM. To measure nitrate, the addition of nitrate reductase and NADPH (20 µl; Boehringer Mannheim, Mannheim, Germany) for 30 min was required for conversion to nitrite. Nitrite was measured by the addition of 100 µl of Greiss reagent to all wells. Trichloroacetic acid (100 µl) was added to precipitate protein. The plate was centrifuged, and OD of each sample was read at 540 nm with a reference wavelength of 650 nm using a microplate reader (Molecular Devices, Menlo Park, CA). Nitrate and nitrite levels were quantified by reading against appropriate standard curves. The results were expressed as micromolar concentrations of RNI, i.e., the sum of both nitrate and nitrite concentrations.
RNI levels determined in CSF and spinal cord tissue
Samples of CSF were obtained from rats following anesthesia. The
rats were shaved over the back of the skull and neck, and a 25-gauge
needle (1 inch minus the hub) attached to 40 cm of 0.5-mm cannula
tubing (Dural Plastics and Engineering, Sydney, Australia)
connected to a 1-ml syringe was inserted 
6 mm behind the
occipital prominence. The dura was penetrated, and the needle passed
the spine of the atlas before entering the cisterna magna. CSF flows
into the tubing, and suction is gently applied using the syringe. The
suction must be released before removing the needle so that blood does
not contaminate the samples. Aliquots of between 20 and 100 µl/ml
were routinely obtained and transferred in duplicate to a V-bottom
immunoassay plate (Nunc) for RNI determination. To correlate RNI data
between the CSF and serum taken from the same animal, the standard
curve for these analyses was made with distilled water.
MBP-specific lymphocyte proliferation assay
Draining lymph nodes and spleens were harvested on day 10 following rechallenge and made into a single-cell suspension by gently passing the node or spleen through a metal sieve (400 mesh) into mixed lymphocyte culture medium supplemented with 1 µl/ml antibiotics. The cells were washed twice and counted. The cell suspension for each sample was diluted in complete mixed lymphocyte culture medium (containing 10% FCS; 1% sodium pyruvate, L-glutamine, and nonessential amino acids; 1 µl/ml antibiotics; and 0.5 µl/ml 2-ME) to give a final cell concentration of 5 x 105/ml. A total of 200 µl cell suspension was added in triplicate to a 96-well round-bottom plate (Nunc). Each sample was incubated with Ag at 5, 10, and 20 µg/ml guinea pig MBP or with no Ag to determine nonspecific proliferation. The cells were incubated for 24 h and 3 and 7 days at 37°C and 5% CO2, then labeled with 5 µCi of [3H]thymidine and incubated overnight. Cell cultures were harvested and radioactivity was determined on a Packard liquid scintillation counter (Packard Instrument, Downers Grove, IL). The stimulation index was calculated as the degree of proliferation of Ag-stimulated cells divided by unstimulated cells from the same sample.
Histological analysis of H&E-stained paraffin sections from rat spinal cord
Spinal cords were removed from rats following perfusion with 30 ml of saline, followed by 60 ml of 10% neutral buffered Formalin, and placed in 10% neutral buffered Formalin for 7-day fixation before paraffin embedding. Cross-sections from the spinal cord were placed side by side in paraffin blocks, and 5-µm serial sections were cut and stained with H&E. To quantify the number of inflammatory lesions between animals, eight sections were cut at four different levels, with 100 µm between levels, through each the lumbar-sacral, thoracic, and cervical areas of the spinal cord. A minimum, therefore, of 96 sections per rat, at 12 different levels of the spinal cord, was assessed in a blinded fashion. A lesion was considered as containing not <10 inflammatory cells.
Immunohistochemistry
Immunohistochemical procedures were performed using an Innogenex IHC kit (San Ramon, CA) following the manufacturer’s instructions. Briefly, paraffin sections on silanized slides were taken to water before immersing slides in citrate buffer (pH 6) at 95°C for 20 min for Ag retrieval. Slides were cooled for 20 min at room temperature, and endogenous peroxidase activity was blocked using 0.3% H2O2 in methanol. Primary Ab, either biotinylated mouse monoclonal anti-rat ED-1 1:100, polyclonal anti-iNOS 1:500, or polyclonal anti-nNOS 1:1000 (Sapphire Biosciences, Sydney, Australia) in TBS (pH 7.6) was added to cover appropriate sections and incubated at room temperature for 1 h, followed by secondary biotinylated anti-rabbit Ab for 1 h at room temperature and HRP-streptavidin conjugate for 30 min. The reaction product was visualized using 3-amino-9-ethyl-carbazole, and each section was counterstained with Mayer’s hematoxylin. ED-1-positive staining was assessed and expressed as the number of positive cells/inflammatory lesion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats were immunized with MBP-CFA and allowed to develop disease
and recover. Thirty-four days after immunization (15 days after
recovery), they were placed on oral L-NMA (15 mM) treatment
in the drinking water and reimmunized with MBP-CFA 24 h later.
Treatment was continued for either 8 or 12 days and then discontinued.
As reported previously, such treatment results in reduced levels of
serum RNI and a secondary clinical disease episode in the majority of
animals within 2–5 days of removal of treatment (18). All
animals were then observed long-term, up to 100 days postrechallenge.
As shown in Fig. 1
, not only did 15 of 16
animals have a second episode of disease, but they all, with one
exception, developed multiple relapses and remissions, e.g., eight
episodes in animal 4, group B. A similar pattern of relapsing remitting
disease was seen in animals treated identically with oral
L-NMA, but not rechallenged with Ag (data not shown).
Rechallenged animals not treated with L-NMA do not develop
disease.
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L-NMA treatment of rats recovered from passive EAE does not induce a second episode of disease
Recipients of 40 x 106 Con A-activated
spleen cells from MBP-CFA-immunized donors developed typical adoptive
EAE with clinical disease onset at days 4 and 5 posttransfer and
recovery by days 10 and11 (data not shown). Such recovered animals do
not spontaneously relapse. However, when actively challenged with
MBP-CFA, the rats develop an early (days 5–8) onset of disease, which
has been interpreted as the persistence and reactivation of some of the
originally transferred cells that had reverted to memory cells
(14, 16). When the recipient rats that had recovered from
passive EAE were placed on oral L-NMA treatment for 12 days
and subsequently followed for 8 wk, there were no second episodes of
disease in any of the animals (0 of 9). The same result was obtained in
repeated experiments using both L-NMA treatment and a more
specific iNOS inhibitor, aminoguanidine. There was no evidence of
subclinical disease when four animals were examined histologically 5
days after removal of L-NMA treatment (data not shown).
Fig. 2 illustrates that the animals in
this group had memory cells capable of being reactivated, as shown by
the early onset of disease following active challenge in a cohort of
rats receiving the same initial donor cell population. One
interpretation of this result is that there is the need for a
peripheral Ag depot to develop a secondary disease following
L-NMA treatment, and suggested the effect of the treatment
may be at the level of proliferation of new effector cells.
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Rats that had recovered from actively induced EAE were placed on
oral L-NMA treatment at day 34 and reimmunized with MBP-CFA
24 h later. Another group of recovered rats received a
rechallenge, but no L-NMA treatment, and a group of naive
rats received a primary immunization only. Ten days after immunization,
draining lymph nodes were taken from each group for proliferation
assays, as described in Materials and Methods. Stimulation
indices for individual animals responding to three different doses of
MBP in vitro are presented in Table I.
These data are representative of repeated proliferation assays. The
proliferation in the L-NMA-treated group was
significantly greater than the untreated group at both 10 and 20 µg
MBP. The untreated rechallenged group did not differ significantly from
animals receiving only the primary immunization 10 days previously.
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In the course of our original work (18), recovered
rats were treated with L-NMA for two different lengths of
time following active reimmunization, and it was noted that the animals
showed clinical signs of disease apparently only after withdrawal of
treatment. To formally test whether this is the case, rats were
immunized with MBP-CFA and allowed to develop disease and recover as
usual. On day 34 after disease induction, all animals were put on 15 mM
L-NMA in the drinking water for 24 h and then
rechallenged with MBP-CFA. Rats were allocated to one of four groups in
which L-NMA treatment was continued for 8, 12, 17, or 21
days and then removed. Another group of naive rats was immunized with
the same inoculum of MBP-CFA to show the challenge inoculum was
encephalitogenic. As shown in Fig. 3, in
all cases animals remained clinically well while receiving
L-NMA and only developed signs of disease 2–4 days after
the drug was removed. Again, these L-NMA-treated animals
all developed relapsing remitting disease.
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We examined the histology of groups of L-NMA-treated
rechallenged animals that were either still on treatment and not
clinically ill or taken off treatment 2–3 days previously and were
clinically ill. Forty sections from three levels of spinal cord,
lumbar-sacral, thoracic, and cervical, were examined and scored from
each animal in a blinded fashion. Fig. 4
shows that the animals still being treated with L-NMA and
showing no clinical signs (group C) had just as many lesions as
clinically ill animals that had treatment withdrawn 2 days before
taking tissue for histology (group A). This was true whether histology
was taken day 8 after rechallenge (L-NMA treatment for 6
days) or day 15 (L-NMA treatment for 13 days) after
rechallenge. There was also no difference in the distribution of the
lesions between the two groups with respect to the level of the spinal
cord involved. Group B in Fig. 4 shows the lesion burden in naive
animals, 8 and 15 days after primary immunization with MBP-CFA.
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Quantitatively, there was no apparent difference with respect to
inflammatory lesions between clinically well, rechallenged rats still
on L-NMA and those taken off treatment that were clinically
ill. This did not, however, preclude a qualitative difference with
respect to cell types involved. Since the macrophage is essential for
clinical expression of disease (21), we examined the
localization of ED-1+ cells in the inflammatory
infiltrates between rechallenged, L-NMA-treated animals
with and without clinical signs. Twelve sections per rat from different
levels of the lower spinal cord were stained for
ED-1+ cells, and positive cells were enumerated
in a blinded fashion. Note was also taken of the overall extent of
lesions between the two groups and again found to be similar. There was
no difference in the extent of involvement of
ED-1+ cells within lesions between the two groups
of animals (Fig. 5), nor was there a
difference in distribution; cells were both perivascular and migrated
into the parenchyma. Six sections from both the cervical and thoracic
cord were also examined, and Fig. 6 shows
the pattern of expression and distribution of
ED-1+ cells. ED-1+ cells
were equally represented in both clinically affected and unaffected
animals at all levels of the spinal cord.
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ED-1+ macrophages were equally abundant in clinically well, rechallenged rats still on L-NMA and those taken off treatment that were clinically ill. We therefore stained immunohistochemically for the presence of iNOS in sections of lower spinal cord from both groups of animals. Six to eight sections, serial to those used for the ED-1+ cells, were examined, and iNOS+ cells were counted. Note was also taken of the extent of inflammation, which again was equally intense in both groups. Both groups of animals had comparable numbers of iNOS+ cells, which were quite minimal, representing no >1–2% of the ED-1+ cells (data not shown).
RNI levels in serum and CSF of rechallenged rats treated with L-NMA and presenting either clinically well or with overt disease
The presence of iNOS does not necessarily translate to the
production of NO. We therefore measured RNI levels, as an indicator of
NO production by NOS, in CSF and serum 8 days after rechallenge (Fig. 7). RNI levels in both the CSF and serum
of rats (n = 4) still on L-NMA
treatment and with no clinical signs of EAE (group C) were 20 µM
on day 8 after rechallenge. Rats (n = 4) that had the
L-NMA treatment terminated on day 6 and had
clinical signs of disease on day 8 (group A) had 3–4 times this amount
(70–80 µM) of RNI in both CSF and serum. As has been previously
reported (17, 18), RNI levels in animals receiving a
primary immunization (group B) have not yet significantly increased at
the time of sampling.
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presents results of an experiment in which RNI levels in the CSF of
Lewis rats were measured during a primary course of EAE with and
without treatment with L-NMA (n = 4).
Despite lowered serum RNI levels vs untreated controls,
L-NMA treatment did not inhibit RNI production in
the CSF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have found that such treated rats not only develop a second episode of disease, but also subsequently develop relapsing remitting disease with up to eight episodes of clinical disease occurring over a period of 3–4 mo (the longest time period examined to date). Two other models of chronic relapsing EAE in the rat have been previously described. Polman et al. (22) reported that low dose cyclosporin A treatment of Lewis rats immunized with spinal cord homogenate plus CFA resulted in a chronic relapsing form of EAE. Understanding the mechanism of relapses in this model is somewhat complicated by the myriad of functions of cyclosporin A ranging from alteration of expression of class II MHC transcriptional activator genes on one hand (23) to osteopenia (24) and hypertrichosis (25) on the other. The other recently reported model is that of protracted, relapsing, and demyelinating disease in dark august rats immunized with syngeneic spinal cord and incomplete Freund’s adjuvant (26). The latter disease results in demyelinating inflammatory lesions mainly in the spinal cord and is associated with Abs to myelin ologodendrocyte glycoprotein and cellular reactivity to the rat MBP peptides 69–88 and 87–101. It is of special interest that the disease should in fact become relapsing since the lack of relapse following the first episode appears due to the production of high levels of NO. One might anticipate that following rechallenge there would also be increased NO levels, and if so, why do relapses occur in this circumstance? An obvious answer is that the primary and relapsing diseases are fundamentally different. In a primary disease, there are two factors limiting it to an acute monophasic disease: specific immune regulation and nonspecific NO production. Inhibiting NO in this case is enough to induce another episode. The second episode of disease can be thought of as a boost to the primary immune response. These animals would possibly have a different specific immune regulatory response that may now allow relapses even in the face of increased (above background) NO levels. We are currently examining the relapsing remitting model with respect to NO levels at various times, pathologic parameters such as demyelination, remyelination, glial reactivity, as well as immunologic parameters such as humoral and cellular reactivity and epitope spreading.
To address the mechanism by which L-NMA treatment may promote relapses, we examined whether animals that had recovered from passive EAE could also be induced to relapse by L-NMA treatment. When rats recover from passive EAE, they show no spontaneous relapses, but when actively immunized with MBP-CFA, they develop clinical disease with onset occurring significantly earlier than in naive rats (14, 16). This has been interpreted as a memory response in which a proportion of the originally transferred encephalitogenic cells persist long-term (27) in the recipient as memory cells. Active immunization then stimulates the expansion of both primary effector cells and resident memory cells, resulting in the more rapid attainment of the threshold number of cells needed to elicit clinically apparent disease. To determine whether animals recovered from passively induced EAE could be made relapsing remitting, they were treated with L-NMA for 12 days and then observed for 2 mo. No relapses or evidence of secondary lesions were observed. Memory cells persisted in these animals, as evidenced by an early onset in actively challenged animals, and these results suggest that there is a need for an Ag/adjuvant depot to drive relapses.
NO has been shown to inhibit macrophage class II expression (28) as well as to have a direct effect on T cell proliferation (29, 30), possibly by preventing activation of Janus kinase (31). RNI levels in serum increase as animals recover from EAE and remain elevated for long periods. Continuing elevated production of NO may prevent renewed expansion of effector cells in response to persistent Ag presence (the depot) through the ability of NO to prevent both Ag presentation and proliferation. Inhibiting NO reverses this effect with subsequent renewed expansion of a new wave of effector cells. We have in fact shown in this study that there is a significant increase in Ag-specific lymphocyte proliferation in L-NMA-treated animals rechallenged with MBP-CFA when compared with untreated animals.
Gold et al. (32) described the enhancement of EAE in Lewis
rats treated with another NOS inhibitor,
L-N-iminoethyl lysine
(L-NIL). However, they were unable to induce disease
recurrence using this agent. The difference between their findings and
ours could of course be due to the fact that L-NIL is more
specific for iNOS than is L-NMA; the latter also inhibits
the endothelial (eNOS) and neuronal (nNOS) enzymes. Dose and timing of
treatment could also account for the different results, and most
importantly, based on the findings presented in this work, we would now
suggest that unless Gold et al. (32) had ceased
L-NIL treatment before termination of the experiment,
clinical disease would not have been seen. We have shown in this work
the necessity for cessation of inhibitor treatment in order for
clinical disease to occur (Fig. 3
).
Histological sections were taken at the same time point following rechallenge from L-NMA-treated animals that were either still receiving L-NMA (and not clinically ill), or that had been taken off treatment 2–3 days previously (and showing clinical signs). We found no difference in the inflammatory lesion burden or in the distribution of lesions within the CNS between these groups. These findings are consistent with the known role of NO in leukocyte adhesion and migration, and with the effects of NOS inhibitors on these parameters (5). The clinically well animals had the same extent of macrophage (ED-1+) involvement in their lesions as did clinically ill animals, and both groups showed the same level of iNOS expression immunohistochemically. We therefore measured RNI levels in the CSF of these two groups and found that despite similar lesion burdens, macrophage numbers, and iNOS expression, the clinically well animals had 3–4 times less RNI in their CSF. This suggested that L-NMA was able to cross the blood-brain barrier in rechallenged animals, where it then inhibited in situ NO generation. The fact that clinical disease was observed only after cessation of L-NMA treatment and a corresponding elevation in RNI levels in the CSF further suggested that NO production in the CNS was required for disease expression.
The clinical findings are inconsistent with our original observations
in which L-NMA treatment administered throughout the course
of primary EAE caused a worsening of clinical disease
(17). In a primary course of EAE, continuous
L-NMA treatment caused an enhanced clinical disease,
whereas continuous L-NMA treatment in rechallenged animals
allows cellular infiltration of the CNS, but prevents the onset of
clinical signs until treatment is ceased. One clear difference in these
two cases is in the levels of RNI found in the CSF of
L-NMA-treated animals. Thus, L-NMA treatment in
rechallenged animals kept RNI levels in the CSF low, while treatment
with the same dose during primary disease did not prevent an
EAE-induced rise in RNI levels in the CNS. The reason for this is not
understood, but it could be that the first episode of EAE compromised
the blood-brain barrier, rendering it more permeable to
L-NMA at the time of rechallenge. Whatever the reason,
clinical signs of disease in primary and second episode experiments
correlated with CNS RNI levels 
70 µM.
These findings support the notion that NO may have a dual function in the regulation of EAE. Its inhibition leads to a more severe disease in a primary course of EAE (17, 18, 32, 33), and as we have now shown, its inhibition can initiate a second episode of inflammatory cell accumulation in the CNS. In contrast, it appears as if the production of NO in the CNS, in excess of normal levels, is required for clinical expression of disease. The mechanism for the latter is not clear, but potential rationales can be envisaged. For example, NO may act as a tissue-damaging free radical directly or combine with the superoxide anion (.O2) to generate peroxynitrite (ONOO-). Peroxynitrite is an oxidant as well as a nitrating agent, and can promote lipid peroxidation as well as nitration of tyrosine residues on proteins (34). Peroxynitrite also degrades to form the highly tissue-damaging hydroxyl radical, and any or all of these downstream molecules of NO could possibly play a role in clinical expression of disease. Recently, others have shown that peroxynitrite is formed early in development of EAE and correlated with clinical disease expression in hyperacute EAE in mice (35). Others have shown that destruction of peroxynitrite by a decomposition catalyst resulted in less severe clinical EAE and demyelination in mice (36). We have been unable to confirm these findings in our model. This could perhaps be due to differences between the mouse and rat models of EAE. We have examined the extent of nitrotyrosine formation in our model and have demonstrated only minimal positive staining throughout the CNS for the presence of nitrotyrosine at peak disease. Therefore, despite the fact that peroxynitrite has been strongly implicated as the pathogenic downstream molecule in mouse EAE (34, 35, 36), there are little comparative data to support the same hypothesis in the rat model.
In summary, our previous findings (17, 18) and those of others (32, 33) suggest that NO has a down-regulatory effect in primary EAE. This effect is probably mediated in the periphery through inhibition of T cell proliferation (29), as well as inhibition of cell adhesion and migration (5). In contrast, the present study has shown that NO may have a role to play in expression of clinical disease. A challenge, therefore, exists in exploiting NO as a potential therapeutic target. A better understanding of the mechanisms involved in the pathogenesis of CNS inflammatory diseases may lead to appropriate therapeutic approaches to the use of NO or inhibitors of its synthesis in treatment of such diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nikki C. O’Brien, Neurosciences Research Unit, Canberra Hospital, P.O. Box 11, Woden, ACT 2606 Australia. E-mail address: Nikki.Obrien{at}anu.edu.au
3 Abbreviations used in this paper: NOS, NO synthase; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; eNOS, endothelial NOS; iNOS, inducible NOS; L-NIL, L-N-iminoethyl lysine; L-NMA, N-methyl-L-arginine acetate; MBP, myelin basic protein; nNOS, neuronal NOS; RNI, reactive nitrogen intermediate.
Received for publication June 4, 2001. Accepted for publication September 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent and IFN--independent induction of apoptosis. J. Immunol. 163:4182.This article has been cited by other articles:
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 K. Rejdak, M. J. Eikelenboom, A. Petzold, E. J. Thompson, Z. Stelmasiak, R. H.C. Lazeron, F. Barkhof, C. H. Polman, B. M.J. Uitdehaag, and G. Giovannoni CSF nitric oxide metabolites are associated with activity and progression of multiple sclerosis Neurology, October 26, 2004; 63(8): 1439 - 1445. [Abstract] [Full Text] [PDF]
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 A. P. Olcott, G. Tocco, J. Tian, D. Zekzer, J. Fukuto, L. Ignarro, and D. L. Kaufman A Salen-Manganese Catalytic Free Radical Scavenger Inhibits Type 1 Diabetes and Islet Allograft Rejection Diabetes, October 1, 2004; 53(10): 2574 - 2580. [Abstract] [Full Text] [PDF]
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 A. Monsonego, J. Imitola, V. Zota, T. Oida, and H. L. Weiner Microglia-Mediated Nitric Oxide Cytotoxicity of T Cells Following Amyloid {beta}-Peptide Presentation to Th1 Cells J. Immunol., September 1, 2003; 171(5): 2216 - 2224. [Abstract] [Full Text] [PDF]
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B. Marchetti, M. C. Morale, J. Brouwer, C. Tirolo, N. Testa, S. Caniglia, N. Barden, S. Amor, P. A. Smith, and C. D. Dijkstra Exposure to a Dysfunctional Glucocorticoid Receptor from Early Embryonic Life Programs the Resistance to Experimental Autoimmune Encephalomyelitis Via Nitric Oxide-Induced Immunosuppression J. Immunol., June 1, 2002; 168(11): 5848 - 5859. [Abstract] [Full Text] [PDF]
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