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Laboratory of Immunobiology, Rega Institute, University of Leuven, Faculty of Medicine, B-3000 Leuven, Belgium
Correspondence: Dr. A. Billiau, Rega Institute, University of Leuven, Laboratory of Immunobiology, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: Alfons.Billiau{at}rega.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-)
membrane receptor (IFN-R KO mice) are more susceptible to
collagen-induced arthritis (CIA) than wild-type mice, indicating that
endogenous IFN- plays a protective role in the pathogenesis of CIA.
In IFN-R KO mice, nitric oxide (NO) production during CIA is
impaired. Because NO is known to exert immunosuppressive and
anti-inflammatory effects in certain model systems, the protective
effect of IFN- might be mediated by NO. Here, we tested in wild-type
mice whether inhibition of NO production by metabolic inhibitors,
aminoguanidine (AG) and L-N-(1-iminoethyl)lysine (L-NIL),
could mimic the ablation of the IFN- receptor. A high-dose regimen
of AG supplied in the drinking water inhibited NO production, disease
development, and anticollagen antibody production but was also
associated with transient body weight loss. At a dose and time regimen
that still inhibited NO production but did not cause body weight loss,
AG failed to affect disease scores. Treatment with L-NIL, which more
specifically than AG affects inducible NO production, caused a slight
increase in anticollagen antibody production although not significantly
affecting disease occurrence. These data indicate that the diminished
capacity of the IFN-R KO mice to produce NO following immunization
with collagen is unlikely to account for their higher susceptibility to
CIA.
Key Words: interferon- • nitric oxide • collagen-induced arthritis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role of interferon (IFN)- in CIA was recently addressed by
studies on DBA/1 mice deficient in expressing the ligand-binding
peptide of the IFN- membrane receptor (IFN-R KO mice). It was
found that these mutant mice are highly susceptible to CIA the disease
was significantly accelerated and the incidence of arthritis was found
to be higher than in wild-type mice [1
, 2
].
A similarly increased susceptibility to CIA was found in wild-type mice
that had been pretreated with IFN- neutralizing antibodies. These
results point to a disease-protective role of endogenous IFN- in the
pathogenesis of CIA. Recently, we reported evidence that this
protective effect derives from inhibition of myelopoiesis, in
particular of reduced generation of Mac-1+ cells
[3
].
IFN- is well-known to be a potent inducer of nitric oxide (NO),
in vitro and in vivo [reviewed in ref. 4
].
IFN--induced NO production has been found to suppress
mitogen-induced T-cell proliferation [5
] and has been
proposed to account for the biological activity of "suppressor"
macrophages [6
]. Furthermore, we recently presented
evidence for a protective role of IFN--induced NO in the
anti-CD3-induced cytokine release syndrome [7
]. In view
of these effects of NO, we investigated whether the IFN--mediated
protection in CIA might be mediated by the production of NO. We
reasoned that if this was the case, treatment of mice with inhibitors
of NO synthase should increase the incidence and/or the severity of
CIA.
In the present study, aminoguanidine (AG) was used, because it has been shown to be an inhibitor of NO synthase (NOS) [8 9 10 ]. The effect of L-N-(1-iminoethyl)lysine (L-NIL), which has recently been described as a more selective iNOS inhibitor [11 ], was also tested in our CIA model. We found that, whereas treatment with these inhibitors did reduce NO production in vivo, it failed to augment disease parameters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain of the IFN-R have been described [12
].
These IFN-R KO mice were back-crossed with DBA/1 wild-type mice for
10 generations to obtain the DBA/1 IFN-R KO mice used in the present
study. All mice were bred in the Experimental Animal Centre of the
University of Leuven. The experiments were performed in 8- to
12-week-old mice, but in each experiment, the mutant and wild-type mice
were age-matched with a 5-day limit. The male and female ratio was kept
between 0.8 and 1.3 in each experimental group.
Induction and clinical assessment of arthritis
Native chicken collagen type II (Elastin Products, Owensville,
MO) was dissolved in 0.05 M acetic acid at 2 mg/ml by stirring
overnight at 6°C and emulsified in an equal volume of complete
Freund’s adjuvant (CFA) containing 1.5 mg/ml heat-killed
Mycobacterium butyricum (Difco, Detroit, MI). Mice were
sensitized with a single 100 µl intradermal injection of the emulsion
at the base of the tail. From day 10 postimmunization, mice were
examined daily for signs of arthritis. The disease severity was
recorded following a scoring system for each limb; 0, normal; 1,
redness and/or swelling in one joint; 2, redness and/or swelling in
more than one joint; 3, redness and/or swelling in the entire paw; and
4, deformity and/or ankylosis.
Measurement of body weight
At regular time points, the weight of the individual animals was
measured. The change in body weight was calculated by comparison with
the initial weight at the beginning of each experiment; i.e., [weight
on indicated time point (=dayx) minus initial weight]/initial weight.
NOS inhibitors and treatment schedule
AG (Sigma Chemical Co., St. Louis, MO) and L-NIL (Bachem,
Bubendorf, Switzerland) were the NOS inhibitors used in this study. AG
and L-NIL were administered in the drinking water, given ad libitum at
doses indicated in Results.
Determination of plasma nitrite levels
NO production was determined by measuring its stable degradation
products, nitrate and nitrite. Nitrate was detected after reduction to
nitrite using nitrate reductase. Total nitrite was then determined
spectrophotometrically, as previously described [13
].
Briefly, blood samples were taken from the orbital sinus and were
collected in tubes containing heparin. Plasma was centrifuged within 30
min after collection. Plasma samples were incubated for 2 h at
37°C in the presence of 0.1 U/ml nitrate reductase, 300 µM
ß-reduced nicotinamide adenine dinucleotide phosphate (NADPH), and 5
µM flavin adenine dinucleotide (FAD). After nitrate reduction, NADPH
was oxidized with 10 U/ml L-lactic dehydrogenase and 10 mM sodium
pyruvate for 30 min at 37°C. Griess’ reagent was added, and
absorbance was measured at 540 nm. All reagents were obtained from
Sigma.
Measurement of serum anti-CII antibodies
Blood samples were taken from the orbital sinus and were allowed
to clot for 1 h at room temperature and at 4°C overnight.
Individual sera were tested for antibodies directed to chicken CII by
enzyme-linked immunosorbent assay (ELISA), as described
[1
]. A serial two-fold dilution series of a purified
standard preparation was included in each experiment to allow for
calculation of the antibody content. The standard material was purified
by affinity chromatography from pooled sera obtained from several
IFN-R KO and wild-type arthritic mice, and contained 0.5 mg/ml
immunoglobulin G (IgG).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R KO mice before and
during CIA developmentR KO and wild-type mice were immunized with CII
in CFA on day 0 and were evaluated for clinical signs of arthritis. As
was the case in our previously reported experiments [1
],
the mutant mice developed signs of arthritis more rapidly and with a
higher frequency than wild-type counterparts. Plasma samples from both
types of mice were collected before and at two time points after
immunization and were analyzed for the presence of total nitrite. The
levels on day 0 were 35.4 ± 6.3 µM (n=3) and
99.6 ± 20.3 µM (n=3) for the IFN-R KO and
wild-type mice, respectively. On days 8 and 57 postimmunization, we
found plasma nitrite levels of 43.6 ± 16.4 µM (n=3)
and 30.2 µM (n=2) for the mutant mice, and 123.8 ±
8.1 µM (n=3) and 110.8 ± 20.8 µM (n=3)
for wild-type animals. Thus, at all time points, the total plasma
nitrite levels were lower in IFN-R KO mice than in corresponding
wild-type ones.
Effect of peroral AG on the course of CIA
To assess whether the increased sensitivity to CIA in IFN-R KO
mice might be related to their decreased production of NO, wild-type
mice were treated with AG, an inhibitor of NO synthase.
In a first experiment, AG was administered in the drinking water at a
dose of 2% (wt/vol); this route of delivery and dose of AG had been
shown to be effective in abrogating NO production in mice infected with
M. tuberculosis [14
]. Wild-type mice not
treated with AG and IFN-R KO mice were included for comparison. All
mice were immunized with CII in CFA and were evaluated daily for signs
of arthritis. The cumulative incidences, nitrite levels, anti-CII
antibody serum levels and body weight are shown in Figure 1
. Arthritis signs started to appear in IFN-R KO mice as early as
day 17, against day 45 in the corresponding wild-types (Fig. 1A)
. Final
cumulative incidences of arthritis in IFN-R KO and wild-type mice
were 70% and 46%, respectively. The increased sensitivity of the
mutant mice was associated with decreased NO production, as evident
from significantly lower nitrite levels at all investigated time points
(Fig. 1B)
. A similar decrease in NO production was observed in
wild-type mice that were treated with the 2% AG regimen. However,
clinical symptoms of arthritis were not detectable at all in these
mice. Because the severity of CIA has been found to depend, in part, on
the extent of the humoral immune response against collagen II
[15
], CII-specific antibodies were analyzed in the sera
of the mice. As evident from the data shown in Figure 1C
, the anti-CII
antibody response was significantly reduced in the wild-type mice that
had received the 2% AG treatment.
|
R KO
and wild-type mice were included. The incidences, plasma nitrite
levels, and anti-CII antibody serum levels are shown in Figure 2
. It can be seen that the NOS inhibitor significantly
down-regulated the plasma nitrite levels (Fig. 2B)
. IFN-R KO mice
developed signs of arthritis from day 17 onward, whereas symptoms of
disease appeared in the wild-type control mice on day 41 after
immunization (Fig. 2A) . The mutant mice reached an incidence of 60%,
against 33% in the wild-type controls. Disease development in 0.5%
AG-treated wild-type animals was indistinguishable from that in
untreated controls [mean day of onset±SEM: 50±2 for the
AG-treated mice (n=10) vs. 47.7 ± 4.4 for the control group
(n=9)]. The humoral response against collagen also remained
unaffected by the treatment (Fig. 2C)
. Thus, in contrast to a dose of
2% AG, a dose of 0.5% could not change the development of arthritis
nor the anti-CII antibody profile, although nitric oxide plasma levels
were clearly inhibited.
|
A final experiment was done to test the effect of high-dose AG treatment using a time schedule that might avoid toxicity. In the previous experiments, body weight loss was most obvious during the first 3 weeks of AG treatment and, thus, coincided with the induction phase of CIA. Reduced food and water intake might have been responsible for the observed effects on CIA. Therefore, in the next experiment, a 2% AG pretreatment was initiated 20 days before immunization to allow the mice to adapt to the treatment. Figure 3 shows body weight, cumulative incidence, nitrite levels, and development of humoral immunity. Determination of plasma nitrite levels indicated that with this altered treatment regimen, NO production during the disease was effectively reduced (Fig. 3C) . Body weight loss was evident only in mice that received AG in the drinking water (Fig. 3A) . However, they had recovered and reached their original body weight after 20 days, i.e., the time point of immunization. Pretreatment with 2% AG did not result in a complete inhibition of CIA, as was the case when treatment was started on day 0 (Fig. 1A) . However, the onset of the disease was still significantly delayed [mean day of onset ± SEM: 53±3 (n=9) vs. 34±2 (n=9) for wild-type controls; P<0.002 by Student’s t-test, comparing AG-treated mice with control]. A possible explanation for this retardation of CIA development might be the effect of AG on anti-CII antibody production. Indeed, the mice that received AG displayed a clearly diminished humoral response against CII on day 33 postimmunization (Fig. 3D) . However, at the end of the experiment (day 70), the level of anti-CII antibody of these mice reached a level comparable with that in controls.
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-inducible NO synthase but also constitutive NO synthases and
certain other enzymes, we tested the effect of a more selective
iNOS-inhibitor, L-NIL. A dose of 0.1% in the drinking water was used,
because in a preliminary experiment (unpublished results), this
appeared to be required to inhibit the production of NO; also, this
regimen was not associated with body weight loss. Three independent experiments were done, the results of which are shown in Figure 4 . NO production in the sera of treated animals was significantly decreased at all investigated time points (Fig. 4A) . The incidence of arthritis in L-NIL-treated mice followed a time course that was not significantly different from that in controls (Fig. 4B) ; if anything, the treated mice developed disease more slowly than the controls but reached the same final incidence [13 out of 28 (46.4%) for the L-NIL-treated mice, against 12 out of 30 (40%) for the controls]. Also, the disease scores of affected animals did not differ (unpublished results). Finally, analysis of the anti-CII antibodies revealed a slight but statistically significant up-regulation (on average 1.7-fold) of the humoral response against CII in the L-NIL-treated mice (Fig. 4C) .
|
resulted in increased disease
manifestations and reduced anti-CII antibody formation, blocking of NO
production by L-NIL had the opposite effects, indicating that
endogenous NO production is unlikely to be involved as a mediator of
the protective effect of endogenous IFN-. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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might explain the protective
effect of endogenous IFN- in certain models of autoimmune disease in
mice. In murine EAE and EAU, blockage of endogenous NO production was
indeed found to be associated with augmented disease manifestation
[19
20
21
22
23
24
25
], suggesting that NO, despite its
well-documented ability to cause cell death and tissue damage, can also
suppress development of autoimmunity and ensuing inflammation [for
review, see ref. 26 ]. The present study focused on CIA, a model of
autoimmune arthritis induced in DBA/1 mice by immunization with CII in
CFA. In this model, ablation of IFN- by administration of
neutralizing antibody or by the gene knock-out approach, has been
reported to augment arthritis incidence and severity [1
,
2
], clearly indicating that, like in experimental
autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis
(EAU), endogenous IFN- plays a protective role. Part of the
underlying mechanism of this effect has been clarified recently by the
demonstration [3
] that an important factor is the
suppression by endogenous IFN- of Mac-1+ leukopoiesis
during the induction phase of CIA. In human bone marrow cultures,
IFN- and NO can suppress colony formation, and pharmacological
blockage of NO can partially prevent the suppressive effect of
IFN- [27
]. Therefore, it seemed a plausible
hypothesis that NO would mediate the protective effect of
IFN- in CIA.
First, it was verified that enhanced CIA occurrence in IFN-R
KO mice was associated with reduced plasma levels of nitrite, the
terminal break-down product of NO. When this was found to be the case,
we tested in wild-type mice whether two inhibitors of NO synthase, AG
and L-NIL, could mimic the effect of IFN-R ablation. AG is known to
inhibit not only the IFN--inducible NO synthase
[8
9
10
] but also constitutive NO synthases
[28
29
30
] and certain other enzymes [reviewed in ref. 31
]. Three parameters were studied: (1) incidence, kinetics, and
severity of CIA; (2) plasma level of NO derivatives; and (3) serum
levels of antibodies against CII. In wild-type mice in which CIA was
induced, 0.5% or 2% AG in the drinking water caused a reduction in
nitrite levels. Contrary to what our hypothesis predicted, AG at the
higher dose (2%) completely blocked occurrence of CIA in these mice,
and the lower (0.5%) dose had no effect. Significantly, however, the
2% regime caused severe but transient body weight loss and reduction
in anti-CII antibody levels, raising concern that the blocking of CIA
was a result of pharmacological effects unrelated to inhibition of NO
synthases. When the 2% AG regime was started earlier so that the mice
had adapted to the treatment and had regained weight by the time of
auto-immunization, the treatment still reduced nitrite levels, as well
as antibody levels and CIA scores. However, the effect was much less
pronounced than when AG treatment was started at the time of
immunization, again strengthening the presumption of an aspecific or
even toxic effect.
The second inhibitor tested, L-NIL, is known to more specifically address inducible NOS [11 , 32 ]. Its administration in the drinking water at 0.1% inhibited circulating NO release, although not causing signs of toxicity. This treatment had little effect on the course of CIA. If anything, it was associated with slightly increased anti-CII antibody levels and resulted in a trend toward delayed disease development.
In summary, our study shows that neither AG nor L-NIL, when
administered in regimens that inhibit circulating NO release without
causing gross physiological changes, mimics the effect of IFN-
ablation on the pathogenesis of CIA. Whereas IFN- ablation was
associated with increased disease signs, treatment with the inhibitors
tended to retard disease appearance. Therefore, we conclude that lower
levels of systemic NO release during CIA in IFN-R KO, as opposed to
wild-type controls, cannot account for the higher disease scores in the
knock-out animals.
It remains possible but unlikely that the NOS inhibitors were less
efficient than the IFN-R knock-out mutation in lowering NO
production levels at certain local sites, and thereby failed to achieve
a comparable effect on critical steps in the pathogenesis, such as the
generation of effector or regulator cells. Therefore, it may be of
interest to see how a knock-out mutation of the iNOS gene affects the
susceptibility of mice to CIA (J. S. Mudgett, personal
communication, reporting no difference between iNOS knock-out and
wild-type mice) [26
]. The role of the decreased anti-CII
antibody response, as a possible factor in the increased CIA
susceptibility of the IFN-R KO mice, could not be clarified by the
results obtained with NOS inhibitors, because one of the inhibitors
(AG) inhibited, whereas the other one (L-NIL) up-regulated the antibody
response. A lack of correlation between humoral reponses and disease
severity has also been noted by others [33
,
34
].
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ACKNOWLEDGEMENTS |
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Received November 10, 1999; revised February 17, 2000; accepted February 18, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptor-deficient mice J. Immunol. 158,5507-5513[Abstract]
receptors J. Immunol. 158,5501-5506[Abstract]
receptor-deficient mice is conditioned by mycobacteria in Freund’s adjuvant and by increased expansion of Mac-1+ myeloid cells J. Immunol. 163,3503-3510: biology and role in pathogenesis Adv. Immunol. 62,61-130[Medline]
receptor-deficient mice are hypersensitive to the anti-CD3-induced cytokine release syndrome and thymocyte apoptosis. Protective role of endogenous nitric oxide J. Immunol. 155,3823-3829[Abstract]
receptor Science 259,1742-1745, nitric oxide, and apoptosis J. Exp. Med. 189,219-230 is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide J. Immunol. 163,5278-5286 and tumor necrosis factor- J. Clin. Invest. 96,1085-1092
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4).
*P < 0.05 by Student’s t-test, comparing
with wild-type control. (C) At indicated time points, four randomly
selected mice were bled and sera were tested for antibodies directed to
chicken CII. Bars represent means ± SEM
(n=4). *P < 0.05 by Student’s
t-test, comparing with wild-type control. (D) Body weight
changes in wild-type mice treated with 2% AG in the drinking water.
2-test in each experiment: 0.956, 0.793, and 0.281;
overall P value by