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Regular Article |
From the Department of Neurology,*
Harvard Medical
School, Beth Israel Deaconess Medical Center, Boston, Massachusetts;
and the Department of Pathology,
Albert
Einstein College of Medicine, Bronx, New York
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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, IL-12, and
interferon (IFN)-
, and reactive oxygen and reactive nitrogen
species. All of these factors have been shown to be elevated in active
MS lesions, and animal models support a role for them in disease
pathogenesis.1,2
The anti-proliferative and/or cytotoxic effects of
nitric oxide (NO) have been associated with the persistent production
of high levels of NO that occurs after the activation of the inducible
form of nitric oxide synthase (iNOS).3
The expression of
this enzyme in various cell types is known to be transcriptionally
regulated and to be activated by a combination of pro-inflammatory
signals such as ligands that activate toll-like receptors and/or
cytokines such as IL-1, tumor necrosis factor-, and interferon-
(IFN-).3
NO by itself demonstrates only weak toxic
activity, but congeners formed by auto-oxidation such as
NO2·,
N2O3, and
S-nitrosothiols enhance its cytotoxic potential. The
toxicity of NO is also greatly enhanced when it combines with
O2- to generate peroxynitrite (ONOO-), an
oxidant capable of damaging lipids, proteins, and DNA.4-6
Detection of nitrotyrosine at inflammatory sites serves as a
biochemical marker for peroxynitrite formation.7,8
Activated macrophages/microglia are a major source of reactive oxygen
intermediates, whereas iNOS has been detected in a wide range of
different cell types.
The factors that lead to the activation of iNOS have been shown to be
both cell-type- and species-specific. So, for example, it is known that
in cells of human origin enhancer elements are located upstream of
-4.7 kb within a 10-kb promoter region that contains four functional
nuclear factor-kappa B (NF-
B) elements8
and two
activatory protein-1 (AP-1) sites.9
This pattern of
cis-element expression contrasts markedly with the murine
iNOS promoter, where only 1.0 kb of 5'-flanking sequence containing two
NF-B sites is required for lipopolysaccharide and cytokine
responsiveness and no AP1 sites are detected.10
These data
strongly suggest that the factors that regulate iNOS expression differ
between mice and humans and this has been supported by studies in which
it has been shown that activation of iNOS in human
monocytes/macrophages is relatively refractory to lipopolysaccharide
and cytokine activation, and comparatively little is known about the
regulation of this enzyme in these cells.11,12
However,
iNOS can be readily induced in human cell types of varying lineages by
a combination of cytokines, and in human fetal astrocytes activation by
IL-1 in combination with IFN- forms a potent inducing
stimulus.13
Because IL-1 and IFN- are known to be up-regulated in active MS
lesions, these data would suggest that astrocytes should be activated
to express iNOS at these sites. Two early reports that examined the
expression of NADPH diaphorase staining as an indicator of NO
production supported a role for astrocytes as a source of NO in MS
lesions,14,15
however, two subsequent studies using a
reverse transcriptase in situ polymerase chain reaction
(PCR) hybridization and/or immunocytochemical approach failed to detect
iNOS in astrocytes, and instead implicated cells of the
monocyte/macrophage lineage.16,17
More recently, a study
of brain biopsies from two acute cases of MS in young adults detected
signal for iNOS in both reactive astrocytes and perivascular
monocytes/macrophages, whereas no signal was found in more chronic MS
cases.18
These data suggest that the extent of lesion
activity may critically affect which cell types express iNOS in the
lesion. To address this possibility in greater detail we have examined
MS lesions of varying activity and age for iNOS expression using a
combination of in situ hybridization for iNOS mRNA and
immunocytochemistry for iNOS protein and nitrotyrosine production. The
results support the conclusion that in active MS lesions multiple cell
types, including astrocytes both within and outside of the lesions,
express both iNOS mRNA and protein whereas the distribution of
peroxynitrite is more restricted.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Tissues were derived from archival autopsy material from the
Clinical Neuropathology Service of the Albert Einstein College of
Medicine or from a brain bank established by Dr C. S. Raine at the
same institution. All tissue collection and use was approved by the
Committee on Clinical Investigation of the Albert Einstein College of
Medicine. Early postmortem tissues (4 to 18 hours) were studied from 12
patients (Tables 1 and 2)
with a clinical diagnosis of primary
progressive (case 2) or secondary progressive MS (cases 3 to 7 and 10
to 13). Case 1 came to autopsy with a diagnosis of progressive
multifocal leukoencephalopathy, but was reclassified as Balo’s
concentric sclerosis after neuropathological examination. Tissues were
either snap-frozen and embedded in OCT medium and stored at -80°C
until use, or processed for conventional paraffin embedding. The
classification of the lesions followed the recommendations of the
recent workshop and were determined after extensive analysis of the
distribution of inflammatory cells, T cells, macrophages, and evidence
of myelin breakdown in these tissues.19
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The presence and distribution of iNOS mRNA in MS lesions was
determined by in situ hybridization using both radioactive
and nonradioactive hybridization procedures. Radioactive in
situ hybridization was performed on two acute MS lesions (cases 1
and 2; Table 1
) using riboprobes that were constructed by PCR using
primers for a 325 bp of iNOS (3'CCG TGA CCC AGA ACC CCG AA and 5' CCA
GCA TCT CCT CCT GGT AG) as described.9
The PCR product was
cloned into the TA vector (Invitrogen) and orientation and nucleotide
sequence of inserts confirmed by automated sequencing. Antisense and
sense probes were transcribed in the presence of
35S-UTP from appropriately cleaved template using
Sp6 or T7 RNA polymerases.
Cryostat sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 20 minutes at room temperature, rinsed in phosphate-buffered saline (PBS), and treated with proteinase-K (1 µg/ml) in 1x PBS for 15 minutes at 37°C. Slides were then rinsed in 1x PBS and treated with 0.1 mol/L triethanolamine, pH 8.0, and 0.25% acetic anhydride for 5 minutes at room temperature. Additional acetic anhydride was added to make a 0.5% solution, and slides were incubated for another 5 minutes. They were then rinsed in 2x standard saline citrate (SSC) for 5 minutes and dehydrated through graded alcohols. Hybridization solution (50% formamide, 0.3 mol/L NaCl, 20 mmol/L NaAc, pH 5.0, 5 mmol/L ethylenediaminetetraacetic acid, 10% dextran sulfate, 2% Denhardt’s solution, 50 mmol/L dithiothreitol, 10 mg/ml yeast t-RNA, and probe at 104 cpm/µl) was heated to 80°C for 2 minutes and then applied to the slides, which were incubated overnight at 42°C. Slides were washed in 4x SSC and 10 mmol/L dithiothreitol three times throughout 1 hour. Slides were then dehydrated in graded alcohols, incubated for 15 minutes at 50°C in 50% formamide, 300 mmol/L NaCl, 200 mmol/L NaAc, 10 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L dithiothreitol, and then washed with 2x SSC. Slides were then treated with RNase A in 20 µg/ml of RNase buffer (0.5 mol/L NaCl, 10 mmol/L Tris-HCl, and 1 mmol/L ethylenediaminetetraacetic acid) for 30 minutes at 37°C and then washed with a large volume (1 L) of the RNase buffer for 30 minutes at 37°C. Slides were then washed with 4 to 5 changes of 2x SSC for 1 hour. Finally, the slides were washed with 0.1x SSC for 15 minutes at 50°C before being dehydrated in graded alcohol washes. Slides were dipped in emulsion and incubated for 14 to 21 days at -80°C before development and counterstaining with hematoxylin. Primary cultures of human fetal astrocytes activated with cytokines as described13 were used as positive and negative controls for the antisense and sense probes respectively.
Nonradioactive in Situ Hybridization
Three MS cases showing acute, chronic active, and chronic silent MS lesions were studied. Anti-sense probes consisted of a 32-base oligonucleotide complimentary to mRNA in the carboxy terminus of iNOS (5'AGA GCG CTG ACA TCT CCA GGC TGC TGG GCT GC) and the 3' PCR primer described above. As a control, sense probes were generated using an oligonucleotide complimentary to that described above (GCA GCC CAG CAG CCT GGA GAT GTC AGC GCT CT) and the 5' PCR primer. These probes were labeled by 3' tailing with digoxigenin-UTP according to the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN).
Paraffin sections were dewaxed and rehydrated through xylene and a series of graded alcohol washes, then treated with 0.2 N HCl for 20 minutes at room temperature, 2x SSC for 15 minutes at 70°C, and proteinase-K 1 µg/ml, 1x PBS) for 15 minutes at 37°C, washed in PBS, and then postfixed with 4% paraformaldehyde. After another wash in PBS, sections were incubated in 0.1 mol/L triethanolamine, pH 8.0, and 0.25% acetic anhydride for 5 minutes at room temperature. Additional acetic anhydride was added for a total of 0.5% in the solution and slides were incubated for another 5 minutes. Slides were rinsed in 2x SSC, rehydrated through graded alcohol, then air-dried. Slides were incubated in hybridization solution (50% deionized formamide, 4x SSC, 1x Denhardt’s solution, 0.5 µg/salmon sperm DNA, 0.25 µg/yeast tRNA solution, and 20% Dextran sulfate for 1 hour before the addition of probe, 200 ng in 30 µl of each oligonucleotide). Sections were incubated with 10 to 15 µl of hybridization solution for 18 to 24 hours at 45°C in a humidified chamber. Slides were rinsed with 2x SSC and then washed for 15 minutes in 2x SSC and 50% formamide at 45°C a total of four times. A final wash of 1x SSC at room temperature was performed for 1 hour. Labeling of sections by probe was detected by immunostaining for digoxigenin with nitroblue tetrazolium as the chromogen according to the manufacturer’s instructions (Boehringer-Mannheim). Sections were counterstained with nuclear Fast Red.
Immunocytochemistry
Paraffin sections were microwaved for 7 minutes in distilled water for antigen retrieval and then incubated with 3% H2O2 for 30 minutes, followed by incubation with 10% goat serum for 1 hour. Rabbit serum raised against the human iNOS carboxy terminal peptide corresponding to amino acids 1135 to 1153 (Santa Cruz Biochemical, Santa Cruz, CA) was then applied at a dilution of 1:100 in 10% normal goat serum for 16 hours at 4°C. The sections were then washed and incubated with biotin-conjugated anti-rabbit IgG (DAKO, Carpinteria, CA) for 30 minutes at room temperature and then processed using the ABC technique and diaminobenzidine as substrate (Pierce, Rockford, IL). The specificity of the reaction was determined by pre-absorption with the specific iNOS peptide (20-fold excess for 2 hours, room temperature; Santa Cruz). Parallel sections were stained for immunoreactivity to glial fibrillary acidic protein (BioGenex, San Ramon, CA), S100ß (DAKO), or CD68 (KP-1; DAKO). For double labeling for iNOS and cell-type-specific markers, sections were first immunostained for iNOS using diaminobenzidine (brown) as the chromogen, followed by incubation with mouse monoclonal antibodies directed against GFAP or CD68 and alkaline-phosphatase-conjugated secondary antibodies for 4 hours room temperature. Nitroblue tetrazolium was used as the chromogen.
Nitrotyrosine Immunocytochemistry
Sections were microwaved for 7 minutes in distilled water for antigen retrieval and then incubated with a rabbit antibody against nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) at 1:200 in 10% normal goat serum in PBS for 16 hours at room temperature. Further incubations with secondary antibody and avidin-biotin complex were performed as above. Diaminobenzidine was used as the chromogen. Positive controls consisted of sections treated with 0.5 mmol/L of tetranitromethane (Sigma Chemical Co., St. Louis, MO) in 50 mmol/L KH2PO2 for 15 minutes, and negative controls using tissues from cases 1 and 2 consisted of sections treated with antibody in the presence of 10 mmol/L nitrotyrosine, as previously described.20
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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In the first set of experiments, we used highly sensitive
radioactive riboprobes on two acute MS lesions to determine whether
iNOS mRNA was present in sufficient abundance to be detected by regular
in situ hybridization procedures, or whether a more
sensitive reverse transcriptase in situ PCR protocol would
be required. As shown in Figure 1
, strong
reactivity for iNOS mRNA was detected in the active lesions. Figure 1A
depicts the edge of an acute lesion with the lesion center to the left
and normal appearing white matter to the right. In the lesion,
prominent accumulations of silver grains were noted decorating large
cells, the size, shape, and distribution of which was consistent with
hypertrophic reactive astrocytes. In addition, an elevated distribution
of silver grains was observed throughout the lesioned area, with the
edge of the lesion clearly demarcated by a drop-off in the intensity of
silver grain deposition. Elevated accumulations of silver grains were
also detected in association with blood vessels both within the lesion
as well as in the adjacent normal appearing white matter (Figure 1, A and C)
. A higher power view of the lesion shown in Figure 1A
depicts
intense labeling of large cells consistent with hypertrophic astrocytes
(arrow) and a lower level reactivity decorating smaller cells in the
lesion (Figure 1B)
. Using a combination of in situ
reactivity for iNOS mRNA and immunoreactivity for CD68, some of these
smaller cells were positive for both (Figure 1B
inset), indicating that
these cells belonged to the macrophage lineage. In Figure 1C, a
higher
power view of an inflamed vessel shows both a linear array of
reactivity, as well as focal accumulations of silver grains, consistent
with reactivity associated with perivascular infiltrates. In contrast,
no silver grains were detected in the same tissues hybridized with a
control sense probe (Figure 1D)
.
|
. When this same lesion was examined at higher
magnification, inflamed vessels within the lesion displayed reactivity
for iNOS mRNA within endothelial cells as well as in some of the
inflammatory cells present in the inflammatory cuffs (Figure 1F)
.
Within the lesion center the large reactive iNOS mRNA reactive cells
displayed the morphology and characteristic distribution of
hypertrophic astrocytes (Figure 1, G and H)
, as illustrated by staining
of parallel sections with S100ß (Figure 1H
, inset). From these data
we conclude that multiple cell types can be activated to express iNOS
mRNA in MS lesions. In contrast to inflamed areas of the CNS, no
reactivity was detected in white matter areas distant from the lesions
(Figure 1I)
. When the acute lesions of the brain were reacted with a
sense probe no reactivity was detected (Figure 1J)
. Chronic-silent MS
lesions showed minimal reactivity (data not shown). iNOS Immunoreactivity in MS Lesions
To examine the distribution of iNOS protein in these same lesions,
the tissues were reacted for immunohistochemistry using a human
iNOS-specific antibody. In previous studies we have shown that this
antibody reacts strongly with primary cultures of human fetal
astrocytes that have been activated by cytokines and that can be shown
to release high levels of NO as determined by the Griess reaction, and
which by Western blot express a single band of 
130
kd.13
In all lesions from seven different cases of MS,
iNOS immunoreactivity was detected within lesioned areas of the brain,
with levels that correlated with lesion activity (Table 1
and Figure 2
). A case of tropical spastic
paraparesis was also studied and showed a moderate degree of iNOS
expression (Table 1)
. Two normal control cases, as well as normal
appearing white matter in MS tissues, showed little or no iNOS
reactivity.
|
. Reactivity associated
with astrocytes was confirmed by double labeling with glial fibrillary
acidic protein (Figure 2, C and D)
. The extent of iNOS reactivity in
macrophages was variable from lesion to lesion, and in some active
lesions perivascular macrophages (as identified by labeling with CD68)
were not stained with the iNOS antibody (Figure 2E)
. In more chronic
lesions, the lesion center was generally nonreactive for iNOS protein,
and iNOS reactivity was localized to scattered reactive astrocytes
along the border of the lesion (Figure 2F)
. The specificity of the
immunoreactivity for iNOS protein was then determined by pre-absorption
of the antibody with the specific peptide supplied by the manufacturer
(Figure 2G)
. iNOS immunoreactivity was also noted in reactive
astrocytes at some distance from the lesion, as well as in association
with noninflamed blood vessels (Figure 2H)
. The expression of iNOS
immunoreactivity in astrocytes at some distance from the lesion has
been noted in the animal model of MS, experimental autoimmune
encephalomyelitis (EAE), and the authors speculate that
astrocyte-derived NO at these sites may be protective and serve to
limit the spread of the inflammatory process.21
This
interesting hypothesis is discussed further in light of the data for
peroxynitrite (see below). Normal appearing white matter remote from
the lesion, as well as white matter from a normal control brain was
nonreactive (Figure 2I)
. IL-1, which has been implicated in the
induction of iNOS in astrocytes,13
was also prominently
expressed in the acute MS lesions (Figure 2J)
. Nitrotyrosine Immunoreactivity in MS Lesions
Seven MS cases were examined immunohistochemically for evidence of
nitrotyrosine, a relatively specific and stable biochemical marker for
peroxynitrite formation, particularly when large amounts are
present.7,8,22
Because the deposition of peroxynitrite
represents a reaction between reactive nitrogen and reactive oxygen
species, its production cannot be localized to one cell but its
presence within the tissues is indicative of the presence of both NO
and O2-. The results are summarized in Table 2
and illustrated in Figure 3
. In acute
lesions intense peroxynitrite staining was detected within the
parenchyma with a distribution pattern suggestive of immunoreactivity
on cell membranes and/or myelin membrane staining, as well as in
perivascular and interstitial locations (Figure 3
; A, B, and C). This
distribution of nitrotyrosine reactivity is remarkably similar to that
recently documented in patients with AIDS dementia
complex,23
and supports previous studies that have
detected the presence of nitrotyrosine in association with disease
activity in the CNS of patients with MS, as well as in
EAE.8,22,24
This close apposition of nitrotyrosine as well
as iNOS immunoreactivity to vessels (illustrated in Figure 3, E and F
)
likely contributes to the damage to the blood brain barrier that is a
cardinal feature of active MS lesions.1
In addition, a
small number of discrete nitrotyrosine-positive cells were detected in
three cases, whereas in two other cases, weak staining was detected in
many cells (Table 2
and Figure 3
). Pre-absorption of the antibody with
nitrotyrosine led to loss of immunoreactivity, including the diffuse
immunoreactivity associated with the lesion center (Figure 3D)
and
inflamed vessels (Figure 3G)
, demonstrating the specificity of the
reaction. Chronic MS lesions (Figure 3H)
as well as normal white matter
in three MS cases and in two normal brains showed no immunoreactivity
(Table 2)
.
|
in the lesion,15
and
our studies further demonstrated prominent reactivity for IL-1 in these
tissues. This distribution of iNOS expression is in general agreement with the notion that NO generated via iNOS may contribute to lesion pathogenesis in MS, but also raises the question as to the function of NO within these tissues.6,25 Although most early reports focused on the potential role of NO as a mediator of tissue injury, after the observation that NO is toxic for oligodendrocytes and neurons,26,27 enhances conduction failure in demyelinated axons,28 and that inhibitors of NO production can protect animals against EAE,29-31 more recent studies have questioned a purely destructive role for NO in these lesions. In particular, data from mice with targeted deletions of the iNOS gene have shown enhanced susceptibility to EAE32,33 and specific inhibitors of iNOS have been found to reactivate EAE in recovered animals,34 suggesting a more significant role for the anti-inflammatory/immunosuppressive properties of NO. Studies of other inflammatory disorders in these mice have also supported an anti-inflammatory role for NO produced via the activation of iNOS.35,36 Nevertheless, it should be kept in mind that the immunosuppressive activities of NO are thought to be mediated principally through the induction of apoptosis in lymphocytes, again attesting to the cytotoxic potential of this free radical.
As noted by Willenborg and colleagues in their recent
review,6
an alternative interpretation is that NO is
generally an immunosuppressive anti-inflammatory molecule whereas in
the target tissues some downstream molecule, such as peroxynitrite, is
responsible for tissue destruction. In the lesions studied here intense
immunoreactivity for nitrotyrosine was detected in most but not all
active MS lesions. This concept is also consistent with the data from
animals with EAE, where widespread evidence of peroxynitrite was
detected in animals with active disease, particularly hyperacute EAE,
but not during disease regression even though the animals still
displayed evidence of inflammation and clinical
activity,22
and the observations that
inhibitors/scavengers of peroxynitrite protect against disease
expression.31,37
Of greater relevance to the current
studies are the data from Cross and colleagues,8
who
demonstrated nitrotyrosine immunoreactivity in active MS lesions and
significantly elevated levels of nitrate (the product of NO and
peroxynitrite) in the cerebrospinal fluid of patients in clinical
relapse. Autopsy CNS tissues from patients with AIDS dementia complex
also showed intense and widespread deposition of nitrotyrosine, which
was not detected in patients who had died with HIV encephalitis not
associated with dementia.24
The authors suggested that the
formation of peroxynitrite in these lesions represented an interaction
between activated macrophages/microglia that functioned as a source of
cytokines such as IL-1 and reactive oxygen intermediates, and
astrocytes that were activated by these cytokines to produce NO. We
would suggest yet a further involvement of activated T cells as a
source of IFN-, a cytokine that is known to act synergistically with
other activators in the sustained production of high levels of iNOS in
many different cell types, including
astrocytes.3,9,10,13,38
From this scenario we predict that
the production of the highly toxic downstream products of NO would
require interactions between appropriately activated lymphocytes,
macrophages/microglia, and astrocytes. Such a complex multiple
signaling pathway requiring cooperative activity between multiple cell
types is encountered frequently in immune-based diseases where the
potential for causing irreversible damage to adjacent uninvolved
tissues constitutes a significant risk. In summary, our results support
a role for multiple cell types, including hypertrophic astrocytes,
macrophages/microglia, and endothelial cells, as sources of NO
production in highly acute MS lesions, whereas as the lesion ages
astrocytes become the more predominant cell type expressing iNOS mRNA
and protein. This would be consistent with the known immunoregulatory
role for astrocytes in CNS inflammation. In addition, the close
proximity of astrocytes to the blood brain barrier and the expression
of iNOS in activated astrocytes as well as peroxynitrite at sites
adjacent to the blood brain barrier also support the possibility that
astrocyte iNOS may contribute to vasodilation and damage to the blood
brain barrier in MS, in addition to immunoregulatory and cytotoxic
roles.
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Acknowledgements |
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Footnotes |
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Supported in part by National Multiple Sclerosis Society grant RG 2771 and United States Public Health Service grants NS 11920, NS 31919, and T32GM07288.
Accepted for publication March 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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. J Neuroimmunol 1993, 46:19-24[Medline]
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M. P. Mycko, R. Papoian, U. Boschert, C. S. Raine, and K. W. Selmaj cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity Brain, May 1, 2003; 126(5): 1048 - 1057. [Abstract] [Full Text] [PDF]
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J. De Keyser, E. Zeinstra, and E. Frohman Are Astrocytes Central Players in the Pathophysiology of Multiple Sclerosis? Arch Neurol, January 1, 2003; 60(1): 132 - 136. [Abstract] [Full Text] [PDF]
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M. Taniike, I. Mohri, N. Eguchi, C. T. Beuckmann, K. Suzuki, a |
