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Department of Microbiology and Immunology, Kimmel Cancer Institute, and the Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
1Correspondence: Department of Microbiology and Immunology, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107-6799, USA. E-mail douglas.c.hooper{at}mail.tju.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: autoimmunity • encephalomyelitis • demyelinating diseases • multiple sclerosis • blood–brain barrier • uric acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Studies
demonstrating that iNOS is expressed and NO.
produced in the spinal cords of mice with experimental allergic
encephalomyelitis (EAE), a model of MS, provide further support for
this concept (3
4
5
6)
. However, NO.,
produced by constitutive forms of NOS, is a mediator involved in the
normal function of both the circulatory and nervous systems (7
, 8)
. In addition, NO. has regulatory
properties that may modulate the autoimmune processes responsible for
EAE (9
, 10)
. These functions of NO.
can be reconciled with a major contribution to toxicity if the
destructive molecule(s) are a product of
NO., for example, peroxynitrite
(ONOO-), rather than NO.
itself. ONOO- is formed by the rapid combination
of NO. and
O2- in a reaction that is
limited only by the diffusion rates of the molecules (11)
.
ONOO- can mediate a variety of destructive
interactions including oxidation (12)
, lipid peroxidation
(11)
, DNA strand breakage (13)
, and nitration
of cysteine and tyrosine residues on proteins (14)
. Thus,
the presence of nitrotyrosine (NT) residues in brain tissues from MS
patients (15
, 16)
and animals with EAE (17
, 18)
is considered to be evidence that
ONOO- may be involved in the pathogenesis of
these diseases. It is also noteworthy that ONOO-
has been associated with pathological changes in other central nervous
system (CNS) disorders including Parkinson’s disease, Alzheimer’s
disease, and amyotrophic lateral sclerosis (19
20
21)
.
To test the concept that ONOO- is involved in
the pathogenesis of CNS inflammatory disease, we have used uric acid
(UA), a natural scavenger of ONOO-
(22)
, to treat EAE (15
, 23)
. Mice, like most
lower mammals, have low serum levels of UA due to its rapid metabolism
by urate oxidase (24
, 25)
to allantoin, which does not
have antioxidant properties (22)
. We previously
demonstrated that the administration of UA to EAE-susceptible mice
immunized with myelin antigens is highly therapeutic, suppressing both
the onset of the disease and ameliorating existing symptoms of EAE
(15
, 23)
. To further our understanding of how
ONOO- may contribute to EAE, we have examined
the effects of UA administration on various parameters of the disease
including blood–CNS barrier permeability as well as the accumulation
and activity of inflammatory cells in the spinal cord.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. UA was administered as a 10 mg suspension
in 100 µl saline intraperitoneally (i.p.) two to four times daily at
intervals of at least 4 h. A minimum of four daily doses of 10 mg
UA are required for a prolonged therapeutic effect in EAE
(23)
. A single 10 mg dose transiently raises serum UA in
mice to levels approaching those seen in human sera as UA is rapidly
metabolized to allantoin in mice by urate oxidase, an enzyme that is
inactive in humans (26
27
28)
. Clinical severity of EAE was
assessed a minimum of twice daily by at least two independent
investigators. Scores were assigned on the basis of the presence of the
following symptoms: 0, normal mouse; 1, piloerection, tail weakness; 2,
tail paralysis; 3, tail paralysis plus hind limb weakness; 4, tail
paralysis plus partial hind limb paralysis; 5, total hind limb
paralysis; 6, hind and forelimb weakness/paralysis; 7, moribund, death
from EAE.
Detection of iNOS, NT, and apoptosis in spinal cord tissue
As spinal cord tissue is the major site of pathology in
MBP-immunized PLSJL mice, spinal cords from UA- and vehicle (saline)
-treated mice were removed and snap frozen in TBS tissue freezing
medium (Triangle Biomedical Sciences, Durham, N.C.) at the times after
immunization indicated in Fig. 1
. The spinal cords were divided into three or four segments and 15 µm
sections were cut in a Lietz cryostat. Approximately every 10th section
of the minimum 100 sections cut per spinal cord segment was assessed
for inflammation by microscopic inspection after Giemsa staining. When
evidence of inflammation was obtained, adjacent sections (both before
and after the Giemsa screened section) were stained as described below.
In control and UA-treated mice where evidence of inflammation in
Giemsa-stained sections was lacking, at least four sets of adjacent
sections from each of the three upper segments of the spinal cord were
stained as described below. iNOS expression was detected in tissue
sections using an iNOS-specific antibody (Santa Cruz Biotechnology,
Santa Cruz, Calif.) as an indicator of inflammation. As nitration of
the ortho position of tyrosine is a major product of
ONOO- attack on proteins (29)
, NT
formation in EAE was assessed using immunohistochemistry with
NT-specific antibodies (Upstate Biotechnology, Lake Placid, N.Y.).
Sections were fixed in 4% paraformaldehyde and stained overnight with
either polyclonal rabbit anti-NT (1/100) or polyclonal rabbit anti-iNOS
(1/200) using the peroxidase antiperoxidase (PAP) method (Sternberger
Monoclonals Inc., Baltimore, Md.) with DAB substrate (brown) and
counterstained with Meyer’s hematoxylin (blue) (30)
. To
determine whether UA treatment modulates the extent of cell apoptosis
seen in EAE, spinal cord sections were assessed for the presence of
cells with fragmented DNA by the TdT-mediated dUTP nick-end labeling
(TUNEL) assay using a commercially available kit (Promega Apoptosis
Detection System, Fluorescein, Promega, Cat. No. G3250, Madison, Wis.)
as detailed in the manufacturer’s protocol (Promega, No. 235). Using
this kit, evidence of apoptosis appears as FITC fluorescence (green)
with viable cells being counterstained with propidium iodide
(red-orange). Photomicrographs were taken with a Sony DKC5000 digital
camera on a Olympus microscope at optical magnifications of
100x (Fig. 2
) and 400x (Fig. 3
).
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Assessment of blood–CNS barrier permeability
To assess the permeability of the blood–CNS barrier to UA, 10
mg UA in suspension was administered i.p. together with 10 mg of the
urate oxidase inhibitor potassium oxonate (K-Ox; oxonic acid, potassium
salt; Acros Organics, Fisher Scientific, Pittsburg, Pa.) to limit the
metabolism of UA during the assay period (31)
. Serum and
tissue UA levels were determined by high-performance liquid
chromatography (HPLC) as detailed elsewhere (32)
. Briefly,
serum was deproteinized by perchloric acid and potassium phosphate
treatment. A 20 µl supernatant sample was injected into a Beckman
System Gold HPLC with a C18 reverse phase column, run at 1 ml/min in a
gradient of 1–35% H2O:acetonitrile:methanol
(50:25:25), and detected at 292 nm. Spinal cord and brain tissues to be
analyzed for UA content were prepared from mice, transcardially
perfused with phosphate-buffered saline (PBS) -heparin, by
homogenization in 9 volumes of 0.4 N perchloric acid, followed by
centrifugation. Excess perchloric acid was neutralized with 25% 1 M
K2HPO4; 20 µl of the
supernatant collected after a second centrifugation was injected into
the HPLC column and analyzed as described for serum UA. UA standards
(0.1 to 10 mg/dl) were run under the same conditions to identify the UA
peak. UA levels were quantitated by integrating the area under the UA
peaks using Gold Nouveau Chromatography Data System Version 1.5
(Beckman Instruments Inc., Fullerton, Calif.). The results of HPLC
analysis of UA levels were confirmed using a quantitative enzymatic
assay (Sigma Chemical Co., St. Louis, Mo., Cat. No. 685–10) according
to the manufacturer’s protocol (Sigma, procedure no. 685).
Blood–CNS barrier permeability was also assessed using a modification
of a previously described technique in which Na-fluorescein is utilized
as a tracer molecule (33
, 34)
. Mice received 100 µl of
10% Na-fluorescein in PBS intravenously under isoflurane anesthesia.
After 10 min to allow circulation of the Na-fluorescein, cardiac blood
was collected and the animals were transcardially perfused with PBS.
Na-fluorescein uptake into the spinal cord was measured using a
modification of the method of Trout et al. (35)
. In brief,
spinal cord tissue was homogenized in 1.5 ml cold 7.5% trichloroacetic
acid and centrifuged for 10 min at 10,000 g to remove
insoluble precipitates. After the addition of 0.25 ml 5N NaOH, the
fluorescence of a 100 µl supernatant sample was determined using a
Cytofluor II fluorimeter (Perseptive Biosystems, Farmingham, Mass.)
with excitation at 485 nm and emission at 530 nm. Serum levels of
Na-fluorescein were assessed as described previously (35)
.
Standards (125 to 4000 µg/µl) were used to calculate the
Na-fluorescein content of the samples in µg. Na-fluorescein uptake
into spinal cord tissue is expressed as [µg fluorescence spinal
cord/mg protein]/[µg fluorescence sera/µl blood] to normalize
values for blood levels of the dye at the time of tissue collection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 23)
. To establish more
conclusively whether UA is therapeutic in EAE through inactivation of
ONOO-, we have assessed the effects of raising
UA levels on several histological parameters relevant to the disease
process, including inflammation, iNOS expression, and NT formation. The
presence in CNS tissue of cells expressing iNOS, the enzyme responsible
for NO. production in an inflammatory response,
has been shown to be a good index of the inflammatory process in
conventional EAE models as well as MS (1
2
3
4
5
6)
. The
expression of iNOS is highly relevant to the generation of
ONOO- as this enzyme is likely to be
responsible, in most forms of EAE, for the production of
NO., which spontaneously combines with
O2- to form
ONOO- (11)
. If UA acts solely by
inactivating ONOO-, raising UA levels should not
have a direct effect on the activation of inflammatory cells, as
evidenced by iNOS expression, but should limit damage specifically
caused by the action of ONOO- in inflamed
tissue. The nitration of tyrosine residues has been the most widely
used indicator of ONOO--mediated tissue damage
in MS and EAE (15
16
17
18)
. We therefore assessed the effect
of the compound, which is known to be a highly effective inhibitor of
tyrosine nitration by ONOO- in vitro
(22)
, on the accumulation of NT residues in spinal cord
tissue from MBP-immunized PLSJL mice. Groups of mice were treated with
UA or vehicle alone, examined daily for clinical signs of EAE (Fig. 1)
,
and their spinal cords were assessed by immunohistochemistry for the
presence of iNOS-positive cells and NT at different intervals after
immunization with MBP (Figs. 2
and 3)
. Four daily doses of 10 mg UA are
the minimum required for the long-term survival of MBP-immunized PLSJL
mice (23)
. One difficulty with the analysis of animals
treated with UA beginning before the expected onset of EAE is that most
do not acquire signs of the disease whereas not all MBP-immunized PLSJL
mice will develop EAE. Animals studied in this investigation were
selected from groups of 36 and 23 mice treated 4x daily with vehicle
and UA, respectively. The incidence of EAE was 72% in the
vehicle-treated group and under 9% in the UA treated group. As a
result, we cannot be sure whether a particular UA-treated animal would
have developed EAE if left untreated, but predict that clinical disease
would have become evident in three of the four UA-treated mice analyzed
here. This is borne out by the fact that 80% of the UA treated mice in
these experiments that were studied after discontinuing 4x daily UA
administration developed severe EAE (clinical score 
5). In
addition to MBP-immunized mice that have remained healthy (ostensibly
because of drug treatment), we have analyzed mice that began UA
administration 4x daily after the clinical signs of EAE had appeared,
as well as animals that developed clinical signs of EAE several days
after drug treatment was reduced from a protective 4x to 2x daily,
which delays the onset of EAE but does not prevent its occurrence
(23)
. Figure 1
details the individual parameters of
treatment and the course of disease for the donors of the
representative spinal cord sections shown in Figs. 3
and 4
. A minimum
of four similar mice were studied for each group. Areas of massive cell
infiltration staining positively for iNOS and NT are evident in
consecutive sections prepared from the spinal cords of MBP-immunized
mice that developed EAE either after UA treatment was reduced from 4x
to 2x daily (Fig. 2C, D
) or in the absence of UA treatment
(Fig. 3A, B
). On the other hand, consecutive sections
obtained from the spinal cord of a mouse that had received UA 4x daily
for 5 days after the appearance of EAE and shown improvement in
clinical signs of the disease exhibit foci of cell infiltration, which
are strongly positive for iNOS but not for NT (Fig. 2E, F
,
Fig. 3D, E
). Spinal cord sections from mice that had been
immunized with MBP, but did not develop clinical signs of EAE while
treated 4x daily with UA, generally resemble those from normal tissue
(Fig. 2A, B
), with little or no evidence of inflammation,
iNOS expression, or NT formation (Fig. 2G, H
, Fig. 3G, H
).
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In addition to the nitration of tyrosine residues,
ONOO- has been linked to the induction of
apoptosis, which, depending on the target cells, may contribute to EAE
pathology (36)
or recovery from the disease
(37)
. We therefore sought to determine whether, in
conjunction with decreasing NT formation in areas of inflammation in
the spinal cord, UA treatment reduces the number of cells staining in
the TUNEL assay for DNA fragmentation, which is associated with
apoptosis and necrosis. As shown in Fig. 3
, this is indeed the case.
Areas of inflammation in the spinal cords of control mice with EAE
contain clusters of cells positive for iNOS, NT, and apoptosis (Fig. 3A-C
). Although iNOS-positive cell accumulation is seen in
spinal cord sections from mice treated 4x daily with UA after disease
development, there is little associated NT or apoptosis (Fig. 3D-F
). Spinal cord sections from an animal treated
with UA 4x daily beginning prior to the onset of EAE showed no
evidence of inflammation, iNOS expression, NT formation, or apoptosis
(Fig. 3G-I
). Analysis of sections from elsewhere in
the spinal cords of these and comparable animals yielded either similar
results or less evidence of histopathology. A summary of our analyses
of spinal cord tissue from a number of 4x daily UA-treated and control
PLSJL mice immunized with MBP is presented in Table 1
. Areas of inflammation positive for iNOS and NT were readily seen in
spinal tissue from animals with acute, progressive EAE. Administration
of UA 4x daily, beginning between 10 and 13 days after MBP
immunization and before the appearance of clinical signs of EAE,
reduced or prevented the appearance of inflammatory cells in the spinal
cord. When begun after the onset of EAE, 4x daily UA administration
selectively interfered with the formation of NT and reduced apoptosis,
evidently without suppressing the ongoing expression of iNOS by
inflammatory cells.
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UA and the blood–CNS barrier in EAE
The blood–CNS barrier, normally reasonably impervious to
UA, is known to be compromised in MS as well as in a rat model of EAE
(38
39
40)
. As shown in Table 2
, i.p. administration of UA leads to its accumulation in spinal cord
tissue of mice with ongoing EAE but not in that of normal controls or
animals in which the disease is no longer progressive. This confirms
that exogenous UA becomes available to inactivate
ONOO- in the spinal cords of mice with
established inflammatory processes. However, the absence of any
significant signs of inflammation in the spinal cords of PLSJL mice
that had begun receiving four daily doses of UA after MBP immunization,
but prior to the onset of clinical signs, suggests that UA may
interfere with the invasion of inflammatory cells into CNS tissue (see
Fig. 2G, H
, Fig. 3G-I
). We speculated
that ONOO- production by cells activated in EAE
may contribute to increasing blood–CNS barrier permeability and hence
to the invasion of inflammatory cells into spinal cord tissue. To test
the hypothesis that ONOO- mediates enhanced
blood–CNS barrier permeability in EAE, MBP-immunized PLSJL with
clinical signs of the disease were treated with vehicle or UA (10 mg)
4x daily for 7 days and microvascular permeability in the spinal cord
was assessed. As shown in Fig. 4
, the blood–CNS barrier leakage that occurs during active EAE is indeed
suppressed by UA administration (Fig. 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 37)
. Superficially, the association between
UA-mediated recovery from EAE and a reduction in the number of
apoptotic cells in spinal cord tissue may support the concept that
apoptosis contributes to the disease process. However, there are other
possibilities and we believe that the results should be taken at face
value: evidence of an association between ONOO-
and the induction of apoptosis in EAE.
The inhibition of NT formation but not iNOS expression in spinal cord
lesions during ongoing EAE implies that UA does not directly interfere
with inflammatory cell activity. Moreover, UA has no effect on the
production of NO. by
lipopolysaccharide-stimulated cells of a macrophage/monocyte cell line
in vitro (23)
. Immunoregulatory mechanisms are
also unlikely to be involved in the mode of action of UA in EAE because
areas of iNOS- and NT- positive cell infiltration in the CNS rapidly
appear, in parallel with clinical signs of EAE, when the minimum
protective UA dose regimen is halved (see Fig. 1
). However, the
appearance of inflammatory foci in spinal cord associated with EAE can
be prevented by UA, providing that treatment is begun at least several
days prior to the expected onset of clinical disease. We speculate that
this is largely a reflection of UA protecting the blood–CNS barrier
from ONOO--induced permeability. Presumably,
ONOO--mediated and therefore UA-suppressible
processes compromise the integrity of the blood–CNS barrier and
thereby provide inflammatory cells, as well as other substances, better
access to the CNS. This concept is supported by our finding that UA
treatment reduced the enhanced permeability of the blood–CNS barrier
to Na-fluorescein normally associated with EAE (Fig. 4)
. Several
previous observations are also consistent with this hypothesis.
Administration of a nonspecific scavenger of reactive oxygen species,
including ONOO-, has been demonstrated to reduce
blood–CNS barrier disruption in experimental optic neuritis
(41)
. ONOO- has also been shown to
increase microvascular permeability in vitro
(42)
. In addition to its effects on the integrity of the
blood–CNS barrier, it is conceivable that ONOO-
may promote cell invasion into the CNS through other means. There is
evidence for an association between the up-regulation of adhesion
molecules, chemokines, and cytokines at the blood–CNS barrier and the
production of reactive oxygen and nitrogen species (reviewed in ref
43
). Further experiments are necessary to fully delineate
the mechanisms through which ONOO- contributes
to CNS inflammation.
The protective role of UA in
ONOO--mediated CNS inflammation may be
particularly relevant to understanding the contribution of elevated
serum UA levels to the evolution of hominoids. In mice, like most lower
animals, UA, a product of purine metabolism, is rapidly oxidized to
allantoin (which does not have antioxidant properties; ref
22
) through the action of urate oxidase (44)
and is normally found at a serum concentration of ~0.5 mg/dl 23. In
humans, however, functional urate oxidase is not present and serum
levels of UA are relatively high, averaging around 4–5 mg/dl in women
and 5–6 mg/dl in men (e.g., ref 32
). The pattern of
mutations responsible for inactivation of the urate oxidase gene in
different primates provides evidence that independent single mutations,
rather than a cumulative stepwise process, were responsible for the
relatively recent acquisition of this inborn error of metabolism
(26
27
28)
. The fact that this error rapidly became dominant
throughout the higher primates has led to the belief that the loss of
urate oxidase must have strong evolutionary advantages (27
, 28)
that far outweigh the contribution of UA to the pathogenesis
of gout (hyperuricemia), where serum UA levels are usually in excess of
9 mg/dl (45
, 46)
. It has been speculated that UA is an
important antioxidant in humans (47)
protecting against
oxidant- and radical-caused aging and cancer (48)
or
oxidative stress in the CNS (49)
. Our finding that raising
serum UA levels in mice to approach those found in normal humans
(23)
is therapeutic in EAE leads us to postulate that the
abrupt evolutionary event that led to the accumulation of UA in higher
primates, despite the threat of UA-caused disease, may be a direct
result of the capacity of this molecule to inactivate
ONOO-. Based on our current findings, we
speculate that serum UA levels in humans, in addition to inhibiting
ONOO--mediated tissue damage, may be important
in maintaining the integrity of the blood–CNS barrier in the face of
circulating cells elaborating ONOO- in response
to a myriad of normal immune stimuli. If so, we believe that low serum
UA levels may predispose an individual toward the development of
MS.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 11, 1999. Revised for publication November 17, 1999.
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