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*
Neurosciences Research Unit, Canberra Hospital; and
John Curtin School of Medical Research, Australian National University, Canberra, Australian Capitol Territory, 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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, and lymphotoxin, whereas their Th2 counterparts
produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are thought to possibly
down-regulate inflammation (5). In addition to the
initiating T cells, recruited macrophages, resident microglia
(6), endothelial cells (7, 8), and astrocytes
(9) all produce their own repertoire of cytokines,
chemokines, and other molecules which may further contribute to the
inflammatory response and to pathology. One putative pathogenic molecule in EAE is NO, produced by the oxidation of arginine in a reaction catalyzed by the enzyme NO synthase (NOS). The inducible form of NOS (iNOS) is up-regulated during inflammation, and this up-regulation can be sustained over a prolonged period of time. An early study demonstrated the increased secretion of reactive nitrogen intermediates (RNI) by inflammatory leukocytes isolated from the CNS and the periphery of rats with hyperacute EAE (10). More recently, increased levels of NO and iNOS mRNA have been localized to the CNS of mice with EAE and correlated with disease severity (11, 12, 13). Furthermore, aminoguanidine, an iNOS inhibitor, has been shown to delay disease onset and decrease severity in murine EAE (14). Thus, NO is often considered a pathogenic molecule. Not all studies on the role of NO in EAE are in agreement however. We (15) and others (16) have shown that treatment of Lewis rats with iNOS inhibitors to prevent NO production enhances actively induced EAE. We further demonstrated that the relatively resistant PVG rat produces up to four times higher serum RNI levels within 48 h of myelin basic protein (MBP)-CFA immunization when compared with Lewis rats and that treating the PVG rats with N-methyl-L-arginine (L-NMA) reduced the NO levels to that of the Lewis rat and rendered them highly susceptible to disease induction. These data strongly suggest that NO can in fact act as a down-regulatory molecule in EAE.
The clinical course of EAE is greatly dependent on the type of Ag, type of immunization (active or passive), species, and strain of animal used to induce disease. In the Lewis rat, EAE actively induced with guinea pig MBP, is an acute monophasic illness from which the rats fully recover. Following recovery from this clinical episode, the rats do not show relapses and within 2 wk of recovery develop a total, long-term resistance to further active reinduction of disease (17, 18, 19). Despite numerous studies, the regulatory mechanisms that determine both the recovery process and the subsequent protection against reinduction of active EAE are not fully understood. Based on our studies with iNOS inhibitors described above (15) indicating a protective role for NO, we advanced the hypothesis that recovery from EAE in the Lewis rat and the subsequent resistance to active reinduction of disease may both be a function of increased production of NO. Here, we report a positive correlation between recovery from disease and the level of circulating RNI. Furthermore, RNI levels not only remain elevated at the time of development of resistance but following rechallenge with MBP-CFA, the levels increase another 2- to 4-fold and remain elevated for up to 2 wk. None of these rechallenged rats develop disease. If rats are treated with L-NMA beginning at peak clinical disease, there is both a prolongation of disease and a change in the presentation of clinical symptoms. Treatment of fully recovered rats with L-NMA 24 h before a rechallenge with MBP-CFA results in a decrease in RNI to below prechallenge levels, and 100% of animals develop a second episode of disease. Remarkably, when recovered rats are treated with L-NMA alone, i.e., in the absence of a rechallenge with Ag, 100% of animals develop a relapse of EAE. These results suggest a central role for NO in the immunoregulation of acute monophasic EAE in the Lewis rat.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Lewis rats (8- to 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 the Animal Holding Facility at The Canberra Hospital. Throughout the experiment, food and water were provided ad libitum, and they were housed under 12-h light and dark cycles.
Induction of EAE
MBP was purified from frozen guinea pig spinal cord according to the method of Eylar et al. (20). Guinea pig MBP in saline was emulsified in an equal volume of ICFA containing added heat-killed Mycobacterium butyricum (4 mg/ml). Rats were anesthetized before immunization with 100 µl of emulsion to each hind footpad for the initial induction of EAE. For rechallenge, rats were immunized with 50 µl of emulsion to each front footpad and 100 µl intradermal in the nuchal region. Total dose received for each immunization per rat was 25 µg of guinea pig MBP and 400 µg M. butyricum. Preliminary studies indicated that both routes of immunization produced EAE with equal incidence, day of onset, severity, and development of resistance.
Evaluation of clinical signs
Rats were examined on a daily basis and clinical scores recorded from day 7 to day 22 postimmunization. Scores were then recorded every other day from day 22 until the time of rechallenge. From rechallenge, they were again examined daily for clinical signs. Clinical disease severity was assessed and scored as described previously (17) using a scale from 1 to 5: 0, asymptomatic; 1, flaccid distal half of tail; 2, entire tail flaccid; 3, ataxia, difficulty in righting; 4, hind limb weakness; and 5, hind limb paralysis.
Histological examination
Rats for study were deeply anesthetized and perfused with 30 ml saline followed by 60 ml 10% neutral buffered formalin. Spinal cords were removed, fixed for 7 days in 10% formalin, and embedded for sectioning. The lumbar-sacral spinal cord was transected, and the halves were embedded side by side for longitudinal sectioning. Six 5-µm sections were cut at various levels through the cord with 50 µm between levels. For quantification, a minimum of 30 sections was counted at different levels.
Inhibition of NO production with L-NMA
L-NMA was prepared using the method outlined by Patthy et al. (21). Lewis rats were housed individually and given L-NMA via their drinking water at times as described in Results. The concentration of L-NMA needed to reduce RNI levels of MBP-CFA-immunized rats to that of unimmunized rats has previously been established as 15 mM/day with the volume of fluid consumed between 15 and 25 ml/rat (15). Because the immunization procedure causes the animals to temporarily reduce their fluid consumption by half, it was necessary to double the concentration of L-NMA in the drinking water for the first 24 h after immunization. The L-NMA solution was prepared daily, filter sterilized, and provided ad libitum to animals housed individually. The daily volume consumed per rat was measured and recorded at the same time each day to ensure delivery of the indicated minimal dose.
Measurement of NO production
The level of nitrate and nitrite in serum samples was determined as an indirect measurement of NO production in vivo as outlined by Rockett et al. (22) and modified and described in detail by Cowden et al. (15). Briefly, 30-µl aliquots of serum was added in duplicate to a V-bottom microplate (Nunc, Roskilde, Denmark). Standard curves were generated using normal dialyzed rat serum to which sodium nitrite or sodium nitrate had been added at concentrations ranging from 1 mM to 1 µM. To measure nitrate, the addition of nitrate reductase and NADPH (20 µl; Boehringer Mannheim, Mannheim, Germany) for 30 min is 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 plates were centrifuged, and the 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 the appropriate standard curves. The results were expressed as micromolar (µM) concentrations of RNI, i.e., the sum of nitrate and nitrite concentrations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats were bled for determination of serum levels of RNI before
immunization on day 0 with 25 µg MBP-CFA and again on days 1, 2, 7,
14, and 21 after immunization. Rats were also assessed for clinical
signs of EAE over this period. Clinical signs first appeared on day 11,
peaked on day 14, and all animals had recovered by day 21 (Fig. 1
). Serum RNI levels increased slightly
24 h after immunization and then remained constant until day 7
when they again began to increase. By day 14 (peak disease), RNI levels
had reached 
8 times background levels and remained elevated as the
animals reached full recovery. These findings are similar to those made
earlier (15).
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One group of rats was immunized with 25 µg MBP-CFA while another
group received no immunization. Both groups were bled for RNI
determination as above, and then on day 35 postimmunization, the
MBP-CFA-treated group was given a second injection of 25 µg MBP-CFA,
and the untreated group was given a primary immunization with the same
inoculum. Rats were again bled on the day of immunization or
rechallenge and on days 1, 2, 7, 14, and 21 postimmunization. There was
an increase in NO production in the MBP-CFA-immunized group when
compared with unimmunized controls (Fig. 2). As before, the increase in serum RNI
remained significantly elevated (p < 0.05)
from day 14 to day 21 after primary immunization, and as shown here
remained elevated out to the time of rechallenge at day 35. After
rechallenge, RNI levels in the MBP-CFA-pretreated group decreased
slightly in the first 24 h and then increased to four times
prerechallenge levels by 48 h and remained increased for 2 wk
before returning to prerechallenge levels. No animal in the MBP-CFA
group displayed clinical signs of disease following the rechallenge,
whereas all animals receiving the primary immunization had severe EAE
(data not shown).
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Rats were immunized with MBP-CFA and developed EAE beginning on
day 9 postimmunization (Fig. 3). By day
11, most rats were severely affected, with clinical scores between 4
and 5. At this time, eight rats were put on oral L-NMA and
the remaining six were maintained on normal drinking water. The
majority of animals in both groups began normal recovery and improved
by at least one clinical score by days 14–15. All untreated animals
completed their rapid recovery and were symptom free by days 15–16 and
remained so for the duration of the experiment. Of the treated animals,
four of eight became symptom free by days 16–17, whereas the other
four maintained mild to moderate clinical signs. On days 18–19, there
was a sudden reappearance of signs in those rats that had recovered and
a rapid worsening in those that had maintained disease. At this time,
all eight L-NMA-treated rats showed the typical pattern of
tail flaccidity and hind limb paresis/paralysis but now also displayed
severe fore limb weakness. One rat became moribund and was killed. The
remaining rats were taken off L-NMA treatment on day 22,
and all recovered by day 27.
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Rats were immunized with MBP-CFA and allowed to develop disease
and recover as normal. On day 35, they were divided into three groups.
One group received normal drinking water and the other two received
L-NMA in the water at either 7.5 or 15 mM beginning 24
h before rechallenge with MBP-CFA. Another group of naive rats was
included to demonstrate the encephalitogenicity of the emulsion used
for the rechallenge. L-NMA treatment was discontinued when
this latter group had all developed clinical signs of EAE, i.e., on day
12. RNI levels were measured, and animals were observed for disease.
Rats that had recovered from active EAE had elevated levels of RNI at
the time of rechallenge as shown previously. Rechallenged nontreated
rats showed a rapid increase in RNI, whereas both
L-NMA-treated groups showed an initial decrease during the
first 48 h followed by a slow increase to the level of the
untreated rats by day 7 (Fig. 4; data
shown only for 15 mM). Most important, 8 of 10 and 6 of 10 rats in the
15 and 7.5 mM L-NMA-treated groups, respectively, developed
a pronounced second clinical episode of EAE (Fig. 5, B and C). No
untreated rechallenged animal developed disease (Fig. 5A),
whereas all of the naive immunized rats developed EAE (Fig. 5D).
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The rats treated with L-NMA that did not develop
clinical signs of EAE (2 at 15 mM and 4 at 7.5 mM L-NMA)
were killed on day 20 after rechallenge, and their spinal cords were
examined for histological evidence of EAE as described in
Materials and Methods. These were compared with three
randomly chosen rats from the untreated rechallenged group and three
from the primary immunized group taken at the same time. As expected,
the primary immunized group showed an extremely heavy lesion burden, on
average 15 lesions/section (Fig. 6).
The untreated rechallenged group showed less than one
lesion/longitudinal section, which were confined mainly to the
meninges. Previous studies from this laboratory (17) have
shown that this level of lesion burden does not differ significantly
from animals receiving only the primary immunization some 55 days
earlier and therefore most likely represents residual lesions from that
immunization rather than new lesions. In comparison, the rats treated
with L-NMA that did not develop clinical EAE,
nonetheless, had extensive lesions throughout the lower spinal cord,
8–10 lesions/section depending on the dose of
L-NMA. These lesions were found throughout the
parenchyma of the white matter as well as in the meninges. The lesions
were qualitatively the same as those seen in animals undergoing primary
disease. The significant difference in the extent and distribution of
these lesions and those seen in the untreated animals suggests that
these must represent new lesions. These data along with the clinical
scores in both of the L-NMA-treated groups
indicates a 100% susceptibility to disease recurrence under such
treatment.
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We next investigated whether treatment of recovered rats with
L-NMA alone without subsequent antigenic rechallenge might
result in a spontaneous relapse of disease. Rats were immunized as
before and allowed to recover from disease. On day 35 postimmunization,
they were put on 15 mM L-NMA in the drinking water for 8
days. Another group was untreated and simply observed for spontaneous
relapses. Remarkably, four of nine L-NMA-treated rats
developed clinical signs of EAE beginning days 10–12 after initiation
of treatment (data not shown). Histology of the lower spinal cord was
examined from the five rats not developing clinical disease and
compared with those from five nontreated rats. All five animals had
extensive lesions when compared with untreated rats (Fig. 7), and the distribution of lesions in
the parenchyma as well as the meninges again indicated new inflammatory
episodes. Thus, 100% of L-NMA-treated rats showed
spontaneous relapses following treatment in the absence of antigenic
rechallenge, whereas none of the untreated rats relapsed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we describe the apparent involvement of NO in this immunoregulation of EAE. Rats recovering from MBP-CFA-induced EAE have significantly increased levels of serum RNI. These levels increase further after rechallenge with MBP-CFA, and all animals are refractory to a second episode of disease. In the experiments reported here, this effect is non-neuroantigen specific in that animals rechallenged with CFA alone show increased RNI levels similar to rats rechallenged with MBP-CFA. In addition, these rats are also refractory to active disease reinduction (data not shown). Oral treatment of rats with L-NMA beginning at peak disease prevents the rapid spontaneous recovery from disease normally seen. Four of eight treated rats did recover briefly but within 2 days had again developed severe clinical signs. This brief recovery may be due to the fact that these rats had temporarily decreased their fluid intake and hence the amount of L-NMA they received. The continued disease in treated rats is remarkable in that all animals exhibit involvement of the fore limbs as well as hind limbs. Acute MBP-CFA-induced EAE in the Lewis rat is an ascending paralysis affecting the tail and hind limbs, and, at least in our hands, very rarely if ever the fore limbs. This observed fore limb involvement could be related to the fact that there are a number of different Ags of the CNS that may become targets of an encephalitogenic T cell response, and the Ag specificity can determine the topography of lesions and hence clinical presentation of disease (37). The change in topography could occur by the reported phenomenon of epitope spreading (38) in which a primary insult to the CNS by T cells of a given specificity results in the generation of new T cells with specificity for other self-determinants either on the same encephalitogenic molecule (intramolecular spreading) or even a different molecule (intermolecular spreading). The possibility of determinant spreading is being investigated.
Treatment of fully recovered rats with L-NMA 24 h before rechallenge with MBP-CFA leads to a rapid decrease in serum RNI levels in these animals and results in a second episode of EAE. An apparent dose-response effect of the L-NMA was seen with 15 mM and 7.5 mM, leading to clinical disease in 80 and 60% of animals, respectively. When histopathological evidence is taken into consideration, i.e., the development of new lesions following challenge as opposed to residual lesions, 100% of L-NMA-treated rats in both groups developed a second episode of EAE. This robust effect was highly reproducible with the same results being obtained in four different experiments.
A recent report by Gold et al. (39) described the enhancement of EAE in Lewis rats treated with L-N6-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS. They also reported the conversion from resistance to susceptibility in L-NIL-treated F-344 rats. We have confirmed both of these results in work done independently using L-NMA as a NOS inhibitor in the Lewis and PVG rat (15). However, Gold et al. (39) reported the inability of L-NIL treatment to eliminate the resistance to reinduction of disease. The reason for the failure to observe reinduction is not known though there are a number of differences between the two studies. One of course is the use of different NOS inhibitors. L-NIL used by Gold et al. (39) is highly selective for iNOS (40), whereas L-NMA inhibits the endothelial (eNOS) and neuronal as well. NO produced in endothelial cells by eNOS affects vascular tone. If, for example, L-NMA significantly reduced eNOS activity, then a mild systemic hypertension may occur (41). We have not measured the blood pressure in our L-NMA-treated rats; however, a link between any mild hypertension and the formation of new inflammatory lesions in the CNS leading to clinical expression of disease would seem somewhat remote.
A more likely explanation for the different results may lie in the
timing and dose of inhibitors given. We have demonstrated that not only
does serum RNI increase during recovery from clinical EAE and remain
elevated to day 35 but when these animals are rechallenged, the
elevated RNI levels double again within the next 24–48 h (Fig. 2
). In
our experiments, treatment with the NOS inhibitor began 24 h
before rechallenge, which resulted in a decrease from these elevated
serum RNI levels back to almost background for the first 48 h. In
the work of Gold et al. (39), there is no mention of the
time of treatment, nor was there any measurement of RNI levels after
rechallenge to determine whether the treatment actually had reduced RNI
levels significantly. Using the same dose of L-NIL as that
used in a rat receiving a primary immunization may not be adequate to
lower RNI levels from an already increased baseline at the time of the
secondary immunization. It is possible therefore that the
L-NIL treatment simply failed to decrease the RNI levels
adequately. We are currently assessing L-NIL in our system
to determine whether the observed difference is related to dose or is
in fact due to inhibitor selectivity.
Treatment of recovered rats with L-NMA alone, without Ag rechallenge, was sufficient to cause a spontaneous relapse of disease in 100% of animals. This effect may be operating at one or two levels. It is known that recovered rats have MBP-reactive T cells, presumably memory cells, which can proliferate in vitro in response to Ag (17, 19) and can also transfer disease to naive recipients (42). MBP-reactive cells have not therefore been deleted in recovered animals, and it is evident that something in the in vivo environment is down-regulating their state of activation. Recovered rats also have an Ag depot which could be the source of stimulus for driving the development of new MBP-reactive precursors; this also is apparently down-regulated since spontaneous relapses normally never occur. NOS inhibition by L-NMA treatment may work by reversing the regulatory mechanism at either or both of these levels. Experiments with passively induced EAE and with removal of the Ag depot will address this question.
Two groups have recently used iNOS knockout mice (iNOS-/-) in the study of the role of NO in EAE, and both reported that average disease severity scores were higher in actively immunized iNOS-/- mice than in wild-type controls (43, 44). Fenyk-Melody et al. (43) also demonstrated that (129SvEv x PL/J)F1 iNOS-/- mice had a greatly decreased ability to recover from disease. Furthermore, aminoguanidine-treated wild-type PL/J mice had an increased incidence and severity of disease and also a decreased remission rate. These authors concluded that iNOS (and by implication NO) may in some instances play a protective role in autoimmune-mediated tissue destruction. The other study also reported an increase in incidence and severity of disease but no evidence for decreased recovery rates in the iNOS-/- animals could be observed because of the chronic nature of the MOG35–55 peptide-induced model employed (44). These authors concluded only that their results did not support the hypothesis that NO is crucial for the development of EAE.
With respect to the mechanism by which down-regulation of NO production by L-NMA treatment allows reinduction or spontaneous relapses of EAE, there are numerous possibilities. NO has a number of known effects on immune responses. It has been shown to inhibit macrophage Ia (45) expression, which would have the effect of preventing T cell expansion due to the lack of Ag presentation. NO is also known to have a direct effect on T cell proliferation (46, 47), probably by preventing activation of Janus kinase (48). This inhibition of T cell proliferation by NO appears in fact to be a specific impairment of Th1 CD4+ T cells while sparing Th2 cells (49). Because EAE is a function of Th1 cells, the increase in NO may selectively limit the proliferation of the encephalitogenic effector population. Also, expression of selectins, VCAM, and ICAM-1 have been found to be down-regulated by NO and hence can significantly alter lymphocyte migration (50, 51). Finally, NO can in some circumstances stimulate the cytokine-mediated release of corticotrophin-releasing factor (52, 53, 54) with resultant production of corticosterone. Corticosterone has been shown to be very effective in down-regulation of EAE (54). Thus, treating with L-NMA and lowering NO levels in recovered animals and preventing an early increase following rechallenge could allow for re-expression of Ia on macrophages and promote Ag presentation and T cell expansion; release both memory and precursor cells from the antiproliferative effect of NO; lead to the re-expression of adhesion molecules and promote migration of effectors into the CNS; and reduce the levels of corticosterone, all of which could promote relapses of disease.
The ability of NO to contribute to recovery from disease may involve nonimmunological as well as immunological mechanisms. In addition to stimulating corticosterone production which, as mentioned, has been shown to promote recovery from EAE (54), NO is known to induce apoptosis or necrosis in T effector cells (13) and to protect oligodendrocytes against destruction by lipid peroxidation (55). Thus, inhibiting these two functions at the level of the target tissue could lead to the lack of recovery and prolonged disease that we observed.
In summary, we have demonstrated that the absence of relapses and the resistance to reinduction of disease seen in Lewis rats immunized with MBP-CFA can be reversed by treatment of rats with oral L-NMA, a specific inhibitor of NOS. It is apparent therefore that although NO can have detrimental effects and contribute to immune-mediated pathology, it can also act in a positive way to down-regulate the immune response. The data presented here sound a cautionary word about the possible use of NOS inhibitors in the therapy of autoimmune disease.
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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:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; iNOS, inducible NO synthase; L-NMA, N-methyl-L-arginine acetate; RNI, reactive nitrogen intermediates; eNOS, endothelial NOS; MBP, myelin basic protein; L-NIL, L-N6-(1-iminoethyl)lysine.
Received for publication July 12, 1999. Accepted for publication October 6, 1999.
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and interleukin-1. J. Immunol. 144:2999.[Abstract]
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