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Clinical and Diagnostic Laboratory Immunology, July 1998, p. 438-445, Vol. 5, No. 4
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Inducible Nitric Oxide Synthase and Nitrotyrosine
Are Found in Monocytes/Macrophages and/or Astrocytes in Acute, but
Not in Chronic, Multiple Sclerosis
Emilia L.
Oleszak,1,2,*
Ewa
Zaczynska,1
Meena
Bhattacharjee,3
Catalin
Butunoi,4
Agustin
Legido,5 and
Christos
D.
Katsetos6
Fels Institute for Cancer Research and Molecular
Biology,1
Departments of Biochemistry
and Neurology,2 and
Department of
Microbiology and Immunology,6 Temple University
School of Medicine, and
Section of Pediatric Neurology, St.
Christopher's Hospital for Children, Allegheny University of the
Health Sciences,5 Philadelphia,
Pennsylvania;
Department of Pathology, Baylor College of
Medicine, Houston, Texas3; and
Rocky
Mountain Multiple Sclerosis Center, Englewood,
Colorado4
Received 13 January 1998/Returned for modification 2 March
1998/Accepted 18 March 1998
 |
ABSTRACT |
We have examined the localization of inducible nitric oxide
synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine
by peroxynitrite, a highly reactive derivative of nitric oxide [NO])
in demyelinating lesions from (i) two young adult patients with acute
multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse
sclerosis), and (iii) five adult patients with chronic MS. Previous
reports have suggested a possible correlation between iNOS,
peroxynitrite, related nitrogen-derived oxidants, and the demyelinating
processes in MS. We have demonstrated iNOS-immunoreactive cells in both
acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS
was localized in both monocytes/macrophages and reactive astrocytes.
However, foamy (myelin-laden) macrophages and the majority of reactive
astrocytes were iNOS negative. In specimens from the childhood MS
patient, iNOS protein was present only in a subpopulation of reactive
or hypertrophic astrocytes. In contrast, no iNOS staining was detected
in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions
with an antibody to nitrotyrosine revealed codistribution of iNOS- and
nitrotyrosine-positive cells, although nitrotyrosine staining was more
widespread in cells of the monocyte/macrophage lineage. In
diffuse-sclerosis-type lesions, nitrotyrosine staining was present in
hypertrophic astrocytes, whereas it was absent in chronic-MS lesions.
These results suggest that NO and nitrogen-derived oxidants may play a
role in the initiation of demyelination in acute-MS lesions but not in
the later phase of the disease.
| |
INTRODUCTION |
Nitric oxide (NO) is a radical
molecule, synthesized by nitric oxide synthase (NOS) from
L-arginine by nitrogen oxidation of guanidino nitrogen to
form L-citrulline (43, 44, 50). There are two
constitutive isoforms of NOS (type I or brain or neuronal NOS and type
III or endothelial NOS) and one inducible form (iNOS or type II)
(9, 15, 16, 43, 51). NO produced by constitutively expressed
NOS (types I and III) plays a major role as regulator and mediator of
numerous processes, including muscle relaxation, vasodilation, and
neurotransmission (43, 44, 50, 51). NO produced by type II
NOS (iNOS) is generated in chronic and acute conditions of inflammation
(9, 15, 16, 19, 26, 30, 34, 48, 52, 64). Type II NOS is
produced by many different cell types in response to endotoxins and
cytokines, such as gamma interferon, interleukin 1, and tumor necrosis
factor alpha (9, 15, 16, 19, 26, 30, 48). Type II NOS has
been detected in several inflammatory diseases of the central nervous
system (CNS), including experimental allergic encephalomyelitis (EAE)
(27) and encephalitis induced by coronavirus, rhabdovirus, flavivirus, rabies virus, Borna virus, herpesvirus, Sindbis virus, and
Theiler's murine encephalomyelitis virus (15, 16, 19, 25-27, 30,
34, 37, 42, 48, 52, 56, 61, 62, 64). Experiments using specific
inhibitors of iNOS revealed that NO may exhibit a protective role in
viral encephalitis by inhibition of viral replication or it may
contribute to the pathogenesis of the disease (7, 17, 37,
42).
It has been reported that iNOS inhibitors may ameliorate EAE in mice
(12, 18, 69). NO produced by microglia could be a potent
neurotoxin and can mediate tumor necrosis factor alpha toxicity towards
oligodendrocytes (20, 47, 49). Therefore, NO produced by
iNOS may be both friend and foe. NO and its degradation products are
reactive molecules and have been implicated in blocking mitochondrial
respiration by forming iron-NO complexes with respiratory enzymes and
enzymes playing a role in DNA replication and repair (40, 66,
67). These results suggest that NO may participate in
demyelinating diseases such as multiple sclerosis (MS), in myelin
damage, or in damage of myelin-producing cells. Dysfunction of
mitochondria may also be the result of formation of peroxynitrite, a
reaction product of NO and superoxide (4, 11, 31, 41, 59).
Peroxidation of membranes as well as swollen oligodendrocyte cell
bodies have been found in the brains of MS patients (29). Peroxynitrite may react with tyrosine in proteins to form nitrotyrosine by adding a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine (5). Nitrosylation of tyrosine has been
observed in cells derived from patients with several acute inflammatory or neurodegenerative diseases, including acute lung injury,
arteriosclerosis, and Alzheimer's disease (5, 24, 35). With
one exception, iNOS expression has been examined only in brain lesions
of chronic-MS patients, and iNOS has been found in active demyelinating
lesions but not in chronic inactive lesions (3, 8, 13, 21,
28). However, there are discrepancies regarding the cell types
that express iNOS. In one study, macrophage/microglial cells have been reported to be the major source of iNOS (3, 21, 28), while in another, astrocytes have been identified as the NO-producing cells
(8, 13). However, NADPH diaphorase staining, which does not
permit the distinction between type I and type II NOS, has been used to
identify iNOS-positive cells in these studies (8, 13).
In this report, we compared the expression of iNOS in the brain in two
cases of acute MS in young adults, one case of diffuse sclerosis in a
child, and five cases of chronic MS. Acute MS represents a distinct
variant of MS and differs both clinically and pathologically from the
much more common classic chronic MS. Acute MS occurs more frequently in
a relatively younger group of patients and is characterized by rapid
and extensive neurological deficit (2, 33, 46, 58). These
patients have multiple, extensive white matter lesions of uniform age
and lack the healed lesions found in chronic-MS patients (2, 33,
46, 58). Diffuse sclerosis represents a rather subacute form of
demyelinating disease which occurs in children and has a relentlessly
progressive clinical course (58). We report here
differential expression of iNOS and nitrotyrosine in brain lesions of
patients with acute and chronic MS.
| |
MATERIALS AND METHODS |
Patients.
The patients described in Table
1 were the source of the
paraffin-embedded specimens used in these studies.
(i) Specimens from acute-MS patients.
Paraffin-embedded
brain biopsy specimens from two patients with acute MS were provided by
the Department of Pathology and Laboratory Medicine, The University of
Texas Health Science Center Medical School of Houston, Tex., and Baylor
College of Medicine, Houston, Tex.
(ii) Specimens from diffuse-cerebral-sclerosis patient.
Paraffin-embedded brain and spinal cord tissues collected at the time
of postmortem from an 11-year-old male with diffuse sclerosis, who died
18 months after diagnosis of the disease, were obtained from St.
Christopher's Hospital for Children, Philadelphia, Pa.
(iii) Specimens from chronic-MS patients.
Paraffin-embedded
autopsy brain tissue was obtained from Rocky Mountain Multiple
Sclerosis Center, Englewood, Colo. (four patients) and Graduate
Hospital, Philadelphia, Pa. (one patient).
Antibodies.
The antibodies employed in these studies were
affinity-purified rabbit anti-iNOS antibody (Transduction Laboratories,
Lexington, Ky.), rabbit anti-glial fibrillary acidic protein
(anti-GFAP) antibody (DAKO, Carpinteria, Calif.), HAM-56 immunoglobulin
M (IgM) monoclonal antibody (MAb) (DAKO), and affinity-purified antinitrotyrosine antibody (Upstate Biotechnology, Lake Placid, N.Y.).
The HAM-56 MAb recognizes an antigen expressed on macrophages, monocytes, and certain microglia (1). We have previously
determined (56) that (i) immunohistochemical staining with
the affinity-purified rabbit anti-iNOS antibody employed in these
studies closely correlates with the presence of iNOS transcripts and
(ii) this affinity-purified rabbit anti-iNOS antibody does not
cross-react with NOS.
Immunohistochemistry.
Five-millimeter-thick paraffin
sections were deparaffinized and rehydrated in xylene and alcohols and
immersed in phosphate-buffered saline (PBS) for 5 to 10 min. Endogenous
peroxidase was blocked by incubation for 30 min in 1.2% hydrogen
peroxide in cold methanol. To eliminate nonspecific staining, sections
were incubated for 1 h with goat serum. iNOS, nitrotyrosine, and
GFAP were detected by an immunoperoxidase technique (54)
using an ABC-ELITE Kit (Vector Laboratories, Burlingame, Calif.).
Sections were incubated with primary antibody, either anti-iNOS (1.25 µg/ml), anti-GFAP antibody (diluted 1:1,000), or antityrosine
(1:200), for 1 h. Antigen-antibody complexes were detected with an
biotinylated anti-rabbit-horseradish peroxidase complex and visualized
with 3,3'-diaminobenzidine as the substrate in accordance with the manufacturer's specifications. Sections were counterstained with Mayer's hematoxylin (Sigma Chemical Co., St. Louis, Mo.). Controls included sections incubated with PBS instead of primary antibody or
sections incubated with normal rabbit IgG (1.25 µg/ml), which was
used instead of a specific rabbit anti-iNOS, anti-GFAP, or antinitrotyrosine primary antibody.
To confirm the specificity of antinitrotyrosine staining,
antinitrotyrosine antibody was preincubated for 1 h with 10 mM
nitrotyrosine in potassium phosphate buffer (pH 7.4) and used to stain
tissue sections. Such treatment totally abolished staining of tissue, which was otherwise positive for nitrotyrosine.
HAM-56-positive cells were detected by using a Universal DAKO LSAB Kit
in accordance with the manufacturer's instruction.
Deparaffinized and
rehydrated tissue sections were digested with
0.025% protease type
XXIV (Sigma) for 6 min at room temperature.
Tissue sections were washed
for 5 min in PBS and blocked for 5
min in blocking buffer before
incubation with HAM-56 MAb. Antigen-antibody
complexes were detected by
sequential incubation of tissue sections
with biotinylated anti-mouse
immunoglobulin and streptavidin conjugated
to horseradish peroxidase.
The reaction was visualized by using
3,3'-diaminobenzidine as the
substrate.
| |
RESULTS |
Expression of iNOS and nitrotyrosine in acute monophasic lesions in
the brains of two patients with acute MS.
For both patients with
acute MS, tissue was procured from surgical biopsy material at the time
of initial clinical presentation. The pathological changes were
essentially similar in both patients and were consistent with a
diagnosis of acute MS. Histologic parameters of these specimens are
shown in Table 2. All lesions were of the
same age, suggestive of a monophasic process. There was no evidence of
shadow plaques in multiple tissue sections examined. Brisk,
predominantly perivascular mononuclear cell infiltrates and
HAM-56-immunoreactive monocytes/macrophages, including foamy macrophages (laden with myelin debris), were admixed with GFAP-positive reactive astrocytes. There was overt myelin loss but preservation of
axons.
Scattered iNOS-labeled cells of the monocyte/macrophage series (Fig.
1a
and b) and reactive
astrocytes (Fig.
1b) were present
within the lesions. The former were
also demonstrated in perivascular
locations (Fig.
1b). The identity of
the two iNOS-positive cell
types was confirmed on subserial sequential
sections immunostained
with anti-HAM-56 (Fig.
1c) or anti-GFAP (Fig.
1d) antibodies,
respectively. It should be noted that iNOS localization
was detected
in only a minority (<10%) of reactive astrocytes, as
well as of
cells of the monocyte/macrophage lineage. Moreover,
virtually
all foamy macrophages were distinctly iNOS negative (Fig.
1a).
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FIG. 1.
Immunocytochemical detection of iNOS protein (a and b),
HAM-56 (c), GFAP (d), and nitrotyrosine (e, f, and g) in active,
monophasic, demyelinating lesions consistent with acute MS. (a)
Evidence of florid demyelinating process characterized by perivascular
inflammatory infiltrates (lymphocytes and monocytes/macrophages),
myelin pallor, and a mixture of mononuclear cells and reactive
astrocytes in the white matter. iNOS localization is present in
scattered cells of the monocyte/macrophage lineage (large arrowheads).
In this field, there is lack of iNOS staining in reactive astrocytes
(small arrows) and in perivascular inflammatory cell cuffs. Original
magnification, ×400. (b) Higher magnification (under oil-immersion
lens) of a field adjoining that of panel a. iNOS-positive cells of
the monocyte/macrophage series (large arrowheads) and somewhat
larger, multipolar cells consistent with reactive astrocytes (small
arrows) are visible. It should be noted that only a minority (<10%)
of mononuclear cells and reactive astrocytes combined are iNOS positive
in these acute lesions. Original magnification, ×1,000. (c) Deeper,
sequential tissue section relative to that of panel a. Widespread
HAM-56 staining of monocytes/macrophages is visible in the perivascular
space and in the white matter. Note morphologic variation among these
cells, including round (monocyte-like), plump (macrophage-like),
rod-shaped, and ameboid (microglia-like) forms. Original magnification,
×400. (d) Homologous microscopic field in a subserial section relative
to sections shown in panels a and c. There is diffuse GFAP staining in
reactive astrocytes and their fibrillary processes, surrounding a
Virchow-Robin (perivascular) space packed with GFAP-negative foamy
(myelin-laden) macrophages. Due to the abundance of macrophages, the
vascular lumen is obliterated. Original magnification, ×400. (e)
Perivascular mononuclear cell cuffing in a white matter blood vessel.
Note scattered monocytes/macrophages within the inflammatory cuff,
showing nitrotyrosine-like immunoreactivity. Original magnification,
×400. (f) Nearby field relative to panel e field. Nitrotyrosine-like
staining is visible in cells of the monocyte/macrophage lineage (large
arrowheads). In this field, reactive astrocytes (small arrows) are
nitrotyrosine negative. During the acute phase of demyelination, the
majority of nitrotyrosine-labeled cells are monocytes/macrophages; only
rare reactive astrocytes are nitrotyrosine positive (not shown).
Original magnification, ×400. (g) Dense perivascular mononuclear cell
infiltrates. As in panel f, nitrotyrosine-like immunoreactivity is
present in scattered monocytes/macrophages. Also, note
nitrotyrosine-positive monocytic elements in the contiguous white
matter (upper right corner). Original magnification, ×400. (h) Deeper
tissue section relative to panel g section. A lack of staining is
apparent in perivascular mononuclear cell infiltrates after the use of
nonspecific IgG in lieu of the primary antibody. Original
magnification, ×400.
|
|
In acute-MS lesions, variable numbers of overt mononuclear cells in the
Virchow-Robin (perivascular) spaces (Fig.
1e and g)
and white matter
(Fig.
1f) exhibited nitrotyrosine-positive staining.
It appears that at
this phase, there is a preponderance of nitrotyrosine
immunoreactivity
in cells of the mononuclear phagocytic series
and a paucity of staining
in reactive astrocytes (Fig.
1f). However,
rare nitrotyrosine-positive
glial cells are also present (data
not shown). As compared to the
cellular localization of iNOS,
the distribution of nitrotyrosine
immunoreactivity in acute-MS
lesions is notably more widespread. The
immunohistochemical findings
for these patients are summarized in Table
3.
Expression of iNOS and nitrotyrosine in specimens from a patient
with diffuse sclerosis.
Brain and spinal cord tissues were
obtained at the time of autopsy of an 11-year-old patient, who died as
a result of a rapidly progressive, unremitting white matter disease
resembling acute MS, with diffuse cerebral and spinal cord involvement,
consistent with so-called diffuse sclerosis (Schilder type). Sections
from multiple gross lesions throughout the neuraxis confirmed a diffuse demyelinating process. All lesions were remarkably of the same age and
exhibited signs of an overt demyelinating process. Extensive multifocal
and confluent lesions characterized by massive accumulations of
HAM-56-positive foamy macrophages (data not shown) imperceptibly merged
with GFAP-positive hypertrophic astrocytes (Fig. 2a and b). Focal and
relatively sparse mononuclear infiltrates were present, but in contrast
to findings in acute-MS lesions, they did not dominate the pathological
lesions. Also, as compared to findings in acute-MS lesions, the
demyelinating lesions in this case contained an increased number of
myelin-laden macrophages, often presenting as extensive tightly packed
accumulations of foamy phagocytic cells (Fig. 2a to c). The histologic
parameters of these specimens are shown in Table 2. Immunohistochemical
staining for iNOS was detected in only a minority (<10%) of
GFAP-positive cells. On the whole, these cells were reactive or
hypertrophic astrocytes (Fig. 2a and c) on the basis of their
morphology and immunoreactivity for GFAP in adjacent sections (Fig.
2b). In addition, robust nitrotyrosine immunoreactivity was present in
these hypertrophic astrocytes (Fig. 2d) but not in foamy macrophages
(Fig. 2d, inset). As with iNOS-positive astrocytes (Fig. 2c, inset), a
predilection for a perivascular location for nitrotyrosine-positive
astrocytes (Fig. 2d) was observed. These immunohistological findings
are summarized in Table 3.
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FIG. 2.
Immunocytochemical detection of iNOS protein (a and c),
GFAP (b), and nitrotyrosine (d) in active, confluent demyelinating
lesions consistent with diffuse sclerosis (Schilder type). (a)
Extensive, confluent demyelinating lesions involving cerebral white
matter. Scattered iNOS-positive reactive or hypertrophic astrocytes are
visible amidst heavy accumulations of iNOS-negative foamy macrophages.
Original magnification, ×100. (b) Subserial section relative to that
of panel a. Accumulations of GFAP-positive hypertrophic astrocytes
intermingled with GFAP-negative foamy macrophages are visible. Original
magnification, ×100. (c) Higher magnification of the microscopic field
depicted in panel a. There is distinctive iNOS immunoreactivity in
large astrocytic cell bodies; however, some degree of staining
variability among morphologically identical hypertrophic astrocytes is
visible. The inset shows discrete iNOS localization in hypertrophic
astrocytes and its processes encroaching on the adventitia of a
parenchymal blood vessel. Original magnification, ×400. (d) Robust
nitrotyrosine-like staining in hypertrophic astrocytes surrounding a
white matter blood vessel. Also, linear-type staining in the luminal
endothelial surface, as well as lack of immunoreactivity in foamy
macrophages (inset), is visible. Original magnification, ×400. (e to
h) Immunocytochemical profile of HAM-56 (e), GFAP (f), iNOS (g), and
nitrotyrosine (h) in subserial sections from a chronic-MS plaque. (e)
Perivascular aggregates of HAM-56-positive monocytes/macrophages
surrounding a sclerotic vessel within a chronic-MS plaque. Original
magnification, ×400. (f) GFAP staining highlights fibrous gliosis
surrounding an intralesional blood vessel. GFAP-negative mononuclear
cells are visible in the perivascular space. Original magnification,
×400. (g) Lack of iNOS staining in perivascular mononuclear cells and
in fibrous astrocytes. Original magnification, ×400. (h) Lack of
nitrotyrosine-like staining in perivascular mononuclear cells and in
fibrous astrocytes mirroring the profile of iNOS. Original
magnification, ×400.
|
|
Expression of iNOS and nitrotyrosine in chronic lesions of classic
MS.
The tissue samples from patients with chronic MS revealed
classical chronic MS plaques involving cerebral hemispheric,
particularly periventricular, white matter. Areas of myelin loss were
accompanied by prominent fibrous astrogliosis, as confirmed by
widespread GFAP staining (Fig. 2f). Focal accumulations of
HAM-56-positive foamy macrophages, exhibiting a predilection for a
perivascular location (Fig. 2e), were present in half of the patients.
Scant, perivascular and/or transmural lymphocytic and
monocyte/macrophage infiltrates were demonstrable in a focal
distribution within chronic plaques in three patients (Fig. 2e and h).
The vascular walls, of presumptive veins, were thickened and
hyalinized, but the vessel lumens were patent (Fig. 2e and h).
Intralesional axons were unaffected. Neither iNOS (Fig. 2g)- nor
nitrotyrosine (Fig. 2h)-positive staining was detected in either
astrocytes or macrophages. Histologic and immunohistochemical findings
for these patients are summarized in Tables 2 and 3, respectively.
| |
DISCUSSION |
NO and NO-dependent oxidants, such as peroxynitrite, are believed
to play a role in the pathogenesis of inflammatory diseases of the CNS,
such as viral and maltose-binding protein-induced encephalitis and
demyelinating diseases, including MS (6, 7, 12, 17, 18, 25, 27,
28, 32, 36, 42, 53, 54, 56, 61, 62, 69). High levels of nitrate,
nitrite, and neopterin, a precursor of tetrahydrobiopterin (a cofactor
for NOS), have been found in the CNS of MS patients (6, 23, 32, 65).
Several mechanisms have been proposed for NO toxicity, including
deamination of DNA, direct inhibition of mitochondrial respiration, and
depletion of NAD by activation of poly(ADP-ribose) synthetase and
formation of peroxynitrite (4, 11, 31, 40, 41, 59, 66, 67).
Peroxynitrite is formed by a diffusion-limited reaction of NO with
superoxide, and it is a powerful oxidant. Membrane lipid peroxidation
of oligodendrocytes has been demonstrated in the brains of patients
with MS (29). Nitration on the ortho position of
tyrosine is the major product of peroxynitrite attack on proteins. NO
does not directly nitrate tyrosine residues. One of the known effects
of tyrosine nitration on protein function is inhibition of
phosphorylation, which is critical in controlling cellular regulation
and signal transduction (45). The presence of nitrotyrosine
in the brains of MS patients suggests the action of peroxynitrite or
other toxic products of NO, such as nitrogen dioxide, in these brains.
Expression of iNOS-derived NO in the brains of MS patients has been
examined by several investigators. However, the cell types that express
iNOS are still a matter of controversy. Bagasra et al. (3)
and De Groot et al. (21) reported that in active MS lesions,
iNOS immunoreactivity was found exclusively in perivascular and
parenchymal macrophages, within the region of active demyelination. Additionally, De Groot et al. (21) demonstrated that
macrophages isolated from active MS lesions produced NO, were stained
with antibody specific to iNOS, and constitutively expressed type I NOS. In contrast, Brosnan et al. (13) and Bö et al.
(8) reported that reactive astrocytes were the major cell
type that expressed iNOS. In the latter study, NADPH diaphorase was
used as a marker for NOS activity, which does not distinguish type I
from type II NOS, suggesting that the NOS detected in astrocytes in
these studies (8, 13) may include type I and/or type II NOS.
However, Brosnan et al. (13) and Bo et al. (8)
did not detect any NADPH diaphorase activity in cells of
monocyte/macrophage lineage. These discrepancies regarding the cell
types that express iNOS in the brains of MS patients prompted us to
examine whether iNOS expression and its cellular localization in the
brains of MS patients is associated with the type of the disease,
namely, acute MS versus chronic MS. We have examined iNOS expression in the brains of two patients with acute MS, one patient with diffuse sclerosis, and five patients with classical chronic MS. In the brains
of patients with acute MS, immunoreactivity for iNOS was found mainly
in perivascular and parenchymal macrophages and in scattered reactive
astrocytes within the demyelinating lesions. In contrast, in the brains
of patients with diffuse sclerosis, which represents a rather subacute,
albeit progressive form of MS in young patients, staining for iNOS was
detected solely in reactive astrocytes. iNOS-positive staining was not
detected in demyelinating lesions of patients with classical chronic
MS. Our results suggest that iNOS expression in acute-MS lesions is
different from iNOS expression in the active demyelinating lesions in
patients with chronic MS described by Bagasra et al. (3), De
Groot et al. (21), and Hooper et al. (28). These
investigators reported (3, 28) iNOS localization exclusively
in cells of the monocyte/macrophage lineage. They did not observe any
iNOS-positive staining of reactive astrocytes. In contrast, in acute-MS
lesions there is iNOS expression in cells of monocyte/macrophages
lineage and in reactive astrocytes. We did not observe iNOS expression
in five patients with classical chronic MS. Our results are in
agreement with those of De Groot et al. (3), who also did
not find any significant iNOS immunoreactivity in demyelinating lesions
in patients with chronic MS. However, diffuse sclerosis might represent
a distinctive form of acute-like MS, since iNOS protein was detected
solely in reactive astrocytes, not in cells of monocyte/macrophage
lineage.
It is of interest that nitrotyrosine-positive staining colocalized with
iNOS immunoreactivity in the brains of two patients with acute MS and
the brain of one patient with diffuse sclerosis, although it was more
widespread than iNOS-positive staining. Tyrosine nitration or iNOS
protein was not detected in patients with chronic MS. Although it has
been suggested that nitration of protein will affect protein stability,
the half-life of nitrotyrosine is unknown. In EAE, expression of iNOS
has been correlated with production of nitrotyrosine in astrocytes in
the spinal cord of mice and reached its maximum at the peak of disease
(53). However, nitrotyrosine-positive cells were eliminated
or degraded 4 to 6 days after the disease was resolved (68).
A similar mechanism may be responsible for the lack of
nitrotyrosine-positive cells in chronic-MS lesions.
The discrepancy between our results and those of De Groot et al.
(21) and Hooper et al. (28) regarding cell types
which express iNOS perhaps may be attributed to the stage of the
disease. Thus, brain tissue examined by Hooper et al. (28)
was obtained at the time of postmortem examination from patients who
had disease for at least 10 years. The age of the patients was not
provided, but these patients had in all probability chronic MS. Among
three brain tissue specimens studied by De Groot et al.
(21), two were from elderly patients (81 years old) and one
was from a 46-year-old patient who also suffered from long-standing
disease. Although the lesions examined by De Groot et al.
(21) and Hooper et al. (28) were characterized as
the result of active demyelinating disease, they are definitely
different from the lesions of the brains of two patients with acute MS
examined in this study. Surgical biopsy specimens from these two
patients were obtained at the time of clinical diagnosis, and they
revealed not only acute but also monophasic lesions, with no evidence
of the secondary progressive demyelinating lesions observed in patients
with chronic MS as reported by De Groot et al. (21) and
Hooper et al. (28). It has been suggested that progression
of demyelinating disease could be the result of epitope spreading, and
lesions at the end stage of the disease may be the result of a
different pathological mechanism than the one that initiated the
disease (39, 63, 68).
It remains to be determined whether expression of iNOS in astrocytes
and monocytes/macrophages in brains of patients with acute MS (this
report) versus expression of iNOS in monocytes/macrophages only in
active lesions of patients with chronic MS (3, 21, 28) can
be attributed to different stages of the same disease or to the
involvement of different pathogenic mechanisms responsible for the
initiation of acute MS. It is well documented, at least in vitro, that
both monocytes/macrophages and astrocytes can produce NO (10, 13,
14, 22, 38, 57, 65).
Our results support the hypothesis that NO and reactive derivatives of
NO, produced by monocytes/macrophages (both perivascular and
parenchymal) and reactive astrocytes, may participate in the demyelination process in acute MS and diffuse sclerosis. These results
are in agreement with the profile of iNOS production in the CNS of mice
infected with Theiler's murine encephalomyelitis virus
(56). Infection of mice with this neurotropic virus results in encephalitis and, later, in a demyelinating disease which resembles MS (reviewed in references 55 and
60). We have found high levels of iNOS transcripts
and protein in astrocytes and monocytes/macrophages in Theiler's
murine encephalomyelitis virus-infected mice during the encephalitic
stage of the disease and the early stage of the demyelinating phase
(56). However, iNOS transcripts and protein were absent
during progression of demyelinating disease (56), which
suggests that NO plays a role in encephalitis and possibly in
initiation of myelin damage but is not involved in progression of the
demyelinating disease.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the N. Eleanor
Naylor Dana Charitable Trust to E.L.O. The Rocky Mountain Multiple Sclerosis Center is a National Multiple Sclerosis funded project.
We thank N. Karmazin and J. P. de Chadarevian, Department of
Anatomic Pathology, St. Christopher's Hospital for Children, Philadelphia, Pa., for providing the CNS specimen from the patient with
diffuse sclerosis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fels Institute
for Cancer Research and Molecular Biology, Temple University School of
Medicine, 3309 North Broad St., Philadelphia, PA 19140. Phone: (215)
707-7657. Fax: (215) 829-1320.
| |
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Abstract
Introduction
