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Short Communications |
B and c-jun Transcription Factors in Multiple Sclerosis Lesions
§
From the Departments of Pathology (Neuropathology),*
Neurology,
and
Neuroscience,§
Albert Einstein College of
Medicine, Bronx, New York; and the Sezione di Neurologia
Clinica,
Dipartimento di Scienze Neurologiche
e della Visione, Università di Verona, Verona, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, are known to generate survival
signals through the activation of the transcription factors NF-
B and
c-jun. The aim of this study was to investigate in
chronic active and silent MS lesions and control white matter the
expression of c-jun, its activating
molecule, JNK, as well as NF-B complex and its
inhibitor, IB. By immunohistochemistry we found negligible
reactivity for these molecules in control white matter and silent MS
plaques. In active MS lesions, double-label
immunohistochemistry with oligodendrocyte markers showed
up-regulation of the nuclear staining for both NF-B and JNK on a
large proportion of oligodendrocytes located at the edge of active
lesions and on microglia/macrophages throughout plaques.
Oligodendrocytes showed no reactivity for IB, which was
predominantly confined to the cytoplasm of microglia/macrophages. We
hypothesize that activation of these transcriptional pathways may be
one mechanism accounting for the paucity of oligodendrocyte apoptosis
reported in MS.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) and interleukin-1 (IL-1)
are of particular interest because, in addition to their
proinflammatory role and apoptotic effect
on oligodendrocytes,4
they are capable of activating
alternative intracellular pathways (viz. the transcription factors
NF-B and c-jun), which may have cytoprotective effects on
target cells.5-10
The transcription factor NF-B is formed by at least five cytoplasmic
proteins (p50, p52, p65 (RelA), c-Rel, and RelB) located in the
cytoplasm of most cells. The complex is physiologically inhibited by
IB molecules; activation of NF-B involves the cytoplasmic
degradation of IB, allowing the translocation of NF-B into the
nucleus where Rel subunits can bind DNA sequences of target genes and
initiate their transcription.6,7
Genes regulated by
NF-B include major histocompatibility proteins, adhesion molecules,
and the cytokines IL-2, IL-6, interferon-ß, transforming growth
factor-ß, and TNF-.7,8
The nuclear protein and
transcription factor c-jun represents a pivotal element in
regulating the neuronal and glial response to injury. Induction of
c-jun and its transcriptional activity involves a molecular
cascade of kinases leading to phosphorylation in the nucleus of
c-jun N-terminal kinases (JNK), which in turn activates its
target, c-jun.9,10
Recent studies have
investigated the expression of NF-B and its inhibitor in MS and in
experimental autoimmune encephalomyelitis on the effector elements of
local inflammation.11,12
However, little is known about
the role of these transcription factors in oligodendrocytes, the
purported target of the autoimmune response in MS.
The aim of this study was to investigate whether the above
transcription factors are involved in oligodendrocyte pathology in MS
lesions. In particular, we examined the expression of molecules
belonging to the NF-B and c-jun pathways to assess
whether their nuclear translocation and activation occurred on
oligodendrocytes within MS lesions, a process that might explain the
reported lack of apoptosis in these glial cells during chronic
MS.13
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early postmortem (4 to 12 hours) CNS tissue was studied from 11 subjects (mean age, 46 years) with a clinical diagnosis of chronic progressive MS. In total, 19 blocks containing lesions and normal appearing white matter were examined. Histopathologically, 4 cases displayed a predominance of chronic active lesions with hypercellular margins, ongoing demyelination with macrophage and lymphocyte infiltration, and an hypocellular demyelinated center with oligodendroglial cell depletion. In the remaining 7 cases, the majority of lesions were defined as chronic silent, based on an absence of inflammation and the presence of demyelinated centers. Brain tissue from 4 subjects (mean age, 57.9 years) with other neurological diseases (OND) was available for control purposes (1 case each of Alzheimer’s disease, stroke, amyotrophic lateral sclerosis, and olivopontocerebellar atrophy). Normal CNS tissue with no evident pathology came from 3 subjects (mean age, 59 years) succumbing to non-neurological conditions. All tissues were snap-frozen and embedded in OCT medium and stored at -80°C until use.
Immunohistochemistry
Frozen sections were air-dried and fixed in cold acetone for 10
minutes. After blocking with normal serum, sections were incubated with
primary antibodies overnight at 4°C. Polyclonal antisera were used to
identify the subunit p65 (C-20) of NF-B and its inhibitor IB
(C-15) (1:1600; Santa Cruz Biotechnology); in addition, a monoclonal
antibody to the activated p65 subunit (ie, recognizing an epitope of
p65 exposed when it is not complexed to IB) was used (1:500;
Boehringer Mannheim). The active, phosphorylated form of JNK was
identified by the monoclonal antibody G-7, recognizing the
pThr-183/pTyr-185 epitope of human JNK1 (1:800; Santa Cruz);
c-jun was identified by the polyclonal antiserum
c-jun/AP-1, recognizing a DNA binding domain of the protein
(1:100; Santa Cruz). Oligodendrocytes were identified with antisera to
the phenotypic markers myelin basic protein (MBP) (1:1000; Dakopatts),
or carbonic anhydrase II (1:800; Chemicon), while resident microglia
and astrocytes were reacted with monoclonal antibodies for CD68 and
glial fibrillary acid protein (Dakopatts), respectively. Appropriate
secondary biotinylated antibodies followed by the avidin-biotin-complex
Elite reagent (Vector Labs) were applied and the reaction visualized
with 3,3'-diaminobenzidine (DAB). For evaluation of the phenotype of
JNK- and NF-B-positive cells, double-staining with an
oligodendrocyte marker was performed. For this, after the peroxidase
immunoreaction for JNK or NF-B was developed with DAB, antiserum to
MBP was applied and detected with an anti-rabbit antibody coupled to
alkaline phosphatase. Red substrate (Vector Labs) was used to visualize
the immunoreaction. Negative controls included omission of the primary
antibody and isotype-specific, irrelevant antibody.
Apoptosis Assay
Apoptotic events in active and silent MS sections were assessed using the terminal deoxynucleotidyl transferase-mediated fluorescein-conjugated deoxyuridine triphosphate nick end-labeling (TUNEL) technique (Boehringer Mannheim), as previously described.13 After fixation with paraformaldehyde and permeabilization with Triton X-100, reaction mixture was added and the samples incubated for 60 minutes at 37°C. The reaction was visualized with an anti-fluorescein Ig conjugated with peroxidase and DAB. As negative controls, the enzyme or secondary antibody was omitted. The phenotype of TUNEL-positive cells was assessed by double immunostaining, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, immunoreactivity
was not seen (Figure 1A)
or showed very
faint staining in scattered astrocytes. Polyclonal antibody to the
inactivated form of p65 gave low level constitutive staining on most
cell types, including oligodendrocytes. In parallel sections, IB was
localized to the cytoplasm of scattered microglia and astrocytes, but
not to oligodendrocytes. Immunostaining for IB and activated JNK and
NF-B subunits in white matter from OND cases was similar to that
observed in normal CNS, while faint c-jun reactivity was
evident on astrocytes (data not shown).
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B reactivity was evident most commonly on hypertrophic astrocytes
which displayed cytoplasmic and, occasionally, nuclear staining (Figure 1B)
. In addition, at the edge of lesions, scattered cells with nuclear
reactivity were present. By double immunohistochemistry with anti-MBP
antiserum, no oligodendrocytes displaying NF-B reactivity were found
(data not shown). Immunoreactivity for c-jun and activated
JNK molecules was similar to that observed for NF-B, while IB
revealed a distinct signal in the cytoplasm of hypertrophic astrocytes
and scattered microglia.
Within chronic active MS lesions and adjacent white matter, both
NF-B and c-jun/JNK reactivity was markedly up-regulated
on glial cells and inflammatory elements (Figure 1, C and D)
.
Staining for the NF-B inhibitor, IB, was detectable in the
cytoplasm of macrophages within plaques and on ramified microglial
cells away from lesions (Figure 1E)
. IB staining was not evident on
cells resembling oligodendrocytes. A positive correlation was observed
between degree of inflammation and extent of glial cells with nuclear
staining for NF-B. As previously described for NF-B
proteins,11
with antibodies for activated NF-B and JNK
subunits, microglia/macrophages were the predominant cell type showing
nuclear reactivity throughout MS plaques. In this study, we focused on
the area encompassing the outer lesion edge and on white matter
immediately adjacent to active lesions where staining for the activated
isoforms of both NF-B and JNK was evident on cells morphologically
resembling oligodendrocytes or microglia, appearances consistent with
chronic signaling in this narrow zone. The pattern obtained for
activated NF-B, c-jun and phosphorylated JNK was similar
both in terms of signal intensity and cellular distribution, and
positively reactive cells displayed a prevalence of nuclear staining
for molecules of the two pathways. With regard to NF-B, results
using a polyclonal antiserum to p65 and a monoclonal antibody
recognizing its active form were comparable. However, the former showed
both cytoplasmic and nuclear staining, while reactivity for the active
form of NF-B was preferentially intranuclear.
Due to the prevalence of nuclear staining of NF-B and JNK proteins,
the identification of reactive cells by purely morphological criteria
was often difficult. For this reason, we performed double
immunohistochemistry with anti-MBP antiserum to identify the phenotype
of cells showing nuclear translocation of NF-B or JNK (Figure 1, F–I)
. At the outer lesion edge and in white matter immediately
adjacent to active MS lesions, about 15 to 30% of cells positive for
either NF-B (Figure 1, F and H)
or JNK (Figure 1, G and I)
gave positive cytoplasmic staining for oligodendrocyte phenotypic
markers and were identified morphologically as oligodendrocytes, while
smaller MBP-negative cells were presumably microglial elements. About
half of the MBP-positive oligodendrocytes around active lesions showed
concomitant nuclear translocation of both NF-B (Figure 1G)
and JNK
(Figure 1I)
.
Regarding the other pathway that TNF- may trigger, ie, cell death by
apoptosis, we confirmed results previously obtained in chronic MS
lesions,13
whereby oligodendrocytes showed no evidence of
DNA fragmentation, while scattered microglial and inflammatory elements
were TUNEL-positive (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and c-jun pathways in
chronic MS lesions to determine whether these transcription factors
might play a role in oligodendrocyte pathology in this purported
autoimmune disease. Activation of both NF-B and c-jun
involves a cascade of intracellular events which lead activated
isoforms in the nucleus to regulate the expression of several genes
involved in cell survival.5-10
By immunohistochemistry,
evidence for up-regulation of both NF-B and c-jun/JNK
signals was found on microglia and oligodendrocytes in chronic active
MS plaques, in comparison to silent MS lesions and control cases (OND
and non-neurological conditions). Signal for both c-jun/JNK
and NF-B proteins was detected in the cytoplasm of astrocytes in
silent MS lesions and control cases, while molecules of both pathways
were up-regulated and localized by double immunohistochemistry in the
nuclei of many oligodendrocytes and microglia at the outer edge of
active MS lesions. No detectable levels of NF-B inhibitor were found
on these cells. In fact, IB was up-regulated in the cytoplasm of
microglia and scattered astrocytes at the edge of active MS lesions and
in the surrounding white matter. Thus, the nuclear pattern for
activated p65 NF-B and JNK molecules, together with the cytoplasmic
reactivity of IB observed in MS lesions, strongly suggested
activation of these transcriptional pathways. Previous studies have
underscored a role for NF-B in MS and its animal model, experimental
autoimmune encephalomyelitis11,12
and in this regard, the
present study confirmed the findings of Gveric et al,11
who showed NF-B activation in resident and infiltrating effector
cells in MS lesions.
Although activation of NF-B and c-jun/JNK pathways in
glial cells may be triggered by a number of
mechanisms,7-10
the correlation observed herein between
extent of transcriptional activation and degree of inflammation in MS
plaques suggested that inflammatory mediators play a major role. Among
elements present within active MS lesions which may trigger these
pathways, IL-1 and TNF- are probably prime candidates, since both
cytokines and their receptors have been documented in MS
plaques.13-16
In this context, investigations in
vitro have shown that TNF- in concert with interferon-
is capable of activating NF-B transcriptional
activity.17
With regard to the pathogenesis of MS,
these observations once again raise intriguing questions about
the role of TNF-, a matter of some debate over recent years. In
fact, both TNF- and its cognate, TNF-ß (or lymphotoxin), are known
to be major inducers of apoptotic cell death in several cell types,
including oligodendrocytes.4,18-20
In normal human CNS
tissue, oligodendrocytes and microglia constitutively express the type
II receptor of TNF-18
and both type I and II receptors
are present on these glial cells in chronic MS plaques.13
Thus, since both ligands and receptors of the TNF system are in place
in MS lesions, one could hypothesize that apoptosis may play a major
role in oligodendrocyte pathology in MS. However, morphological
evidence of significant oligodendrocyte apoptosis has not yet been
found in MS,1,21
and search for DNA fragmentation in these
cells has yielded conflicting results.2,13,22,23
Therefore, although oligodendrocytes display pro-apoptotic receptors
and are susceptible to TNF-mediated apoptotic death in
vitro, there is no compelling evidence that they are depleted
through apoptosis in MS. Several studies have demonstrated that, in
addition to cytotoxicity, TNF- can exert anti-apoptotic activity and
can even prevent apoptosis through the NF-B
complex.24-26
Overall, these observations support the
dual ability of the TNF system to induce either apoptotic cell death or
survival signals through the NF-B pathway. In this context, our
results appear to indicate that the MS plaque microenvironment is able
to activate the NF-B pathway in oligodendrocytes and microglia. We
propose that in concert with other protective mechanisms such as
bcl-213
and ciliary neurotrophic factor,19,20
activation of NF-B may exert anti-apoptotic effects and contribute
to the absence of an apoptotic response by oligodendrocytes in MS.
As for the other TNF-inducible transcription factor, c-jun
is known to play a critical role in neuronal and glial responses to
injury,10
although its final effect on glial cell fate
remains controversial, with some investigators showing that induction
of c-jun by nerve growth factor or TNF- in
oligodendrocytes correlates with apoptosis,27,28
and
others demonstrating that activation of c-jun by TNF- has
no effect on the apoptotic process.5
Clearly, further
experiments are needed to address the precise role of
c-jun/JNK in oligodendrocyte biology. In the present study,
we have found up-regulation of c-jun together with high
signals for activated JNK in the nuclei of oligodendrocytes in chronic
active MS lesions. The concomitant absence of oligodendroglial cell
death would speak against a direct role of c-jun in the
apoptotic process of these glial cells. In this regard, activation of
JNK without apoptosis by TNF- has been reported in astrocyte and
oligodendrocyte cultures.29
Taken in concert, we propose that up-regulation of the transcription
factor NF-B, and possibly c-jun, may help to explain the
lack of convincing evidence of oligodendrocyte apoptosis in chronic MS
lesions, despite the presence of proapoptotic molecules.
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Acknowledgements |
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Footnotes |
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Supported in part by the Fondazione Italiana Sclerosi Multipla (97/R/17) to B.B., and the following to C.S.R.: NS 08952 and NS 11920 from the NINDS; NMSS RG 1001-I-9 from the National Multiple Sclerosis Society; the Sol Goldman Charitable Trust; and the Wollowick Family Foundation.
Accepted for publication July 21, 1999.
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References |
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B activation prevents cell death. Cell 1996, 87:565-576[Medline]
B, and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol 1993, 143:1-62[Medline]
B: ten years later. Cell 1996, 87:13-20[Medline]
B: a crucial transcription factor for glial and neuronal cell function. Trends Neurol Sci 1997, 20:252-258[Medline]
B and inhibitor IB are localized in macrophage in active MS lesions. J Neuropathol Exp Neurol 1998, 57:168-178[Medline]
B is activated in microglia during experimental encephalomyelitis. J Neuroimmunol 1994, 55:99-106[Medline]
and tumor necrosis factor in oligodendrocytes. Eur J Neurosci 1998, 10:2975-2983[Medline]
in human immunodeficiency virus type-1 encephalopathy. Ann Neurol 1995, 37:381-394[Medline]
B in preventing TNF-induced cell death. Science 1996, 274:782-784B. Science 1996, 274:784-787-induced apoptosis by NFB. Science 1996, 274:787-789This article has been cited by other articles:
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 N. Merabova, D. Kaniowska, R. Kaminski, S. L. Deshmane, M. K. White, S. Amini, A. Darbinyan, and K. Khalili JC Virus Agnoprotein Inhibits In Vitro Differentiation of Oligodendrocytes and Promotes Apoptosis J. Virol., February 1, 2008; 82(3): 1558 - 1569. [Abstract] [Full Text] [PDF]
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