Increase of S-100 Immunoreactivity in the Urinary Bladder
from Patients with Multiple Sclerosis, An Indication of
Peripheral Neuronal Lesion
JIANG GU, M.D., JULIA M. POLAK, D.SC, M.D., MRCPATH, ANTONY DEANE, F.R.C.S.,
DOMENICO COCCHIA, M.D., AND FABRIZIO MICHETTI, M.D.
The Schwann cells in urinary bladder biopsies from multiple
sclerosis patients and controls were examined by immunocytochemistry with an antiserum to S-100. S-100 immunoreactivity
was found to be markedly increased in these tissues as compared
with the controls, indicating a Schwann cell hyperplasia in the
urinary bladder in multiple sclerosis. This finding suggests
that local neuronal damage exists in the urinary bladder of
patients with multiple sclerosis. Therefore, the concept of
multiple sclerosis as a disease wholly of the central nervous
system should be reexamined. (Key words: Immunocytochemistry; S-100; Schwann cells; Urinary bladder) Am J Clin Pathol
1984; 82: 649-654
MULTIPLE SCLEROSIS is known as a disease of the
central nervous system, with lesions primarily found in
the white matter of the brain and the spinal cord. The
etiology of multiple sclerosis is uncertain, and specific
therapy is not available. There is some evidence, however,
to suggest that a possible cause may be a virus infection
combined with a dysfunctional immune system. Associated with multiple sclerosis are a number of peripheral
symptoms such as those of the urinary bladder and the
gastrointestinal tract; all of these have been attributed
to lesions in the central nervous system, presumably
being caused by damage in the central neural control of
the affected organ. No primary peripheral morphological
changes have been reported in multiple sclerosis patients.
S-100 is an acidic protein originally extracted from
bovine brain. Its name is derived from its solubility in
100% saturated ammonium sulfate at neutral pH. 33 S100 first was thought to be present exclusively in the
glial elements of the brain including astrocytes, oligodendrocytes, and ependymal ce n s . 5618 - 2627 . 31 Later, it
was found that it also occurs in a number of peripheral
cells including Schwann cells of the peripheral nerves,7,45
Received February 20, 1984; received revised manuscript and accepted
for publication April 24, 1984.
Dr. Gu is a visiting colleague from the Department of Pathology,
Peking Medical College, Peking, China.
The present study was funded by the Medical Research Council and
the Multiple Sclerosis Society of Great Britain.
Address reprint requests to Dr. Polak: Department of Histochemistry,
Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane
Road, London W12 OHS, United Kingdom.
649
Department of Histochemistry, Royal Postgraduate Medical
School, Hammersmith Hospital, Institute of Urology,
Shaftesbury Hospital, London, United Kingdom and
Department of Anatomy, Universita Carrolica, Rome, Italy
satellite cells of the sympathetic ganglia and the adrenal
medulla,7 reticular cells of the lymphoid organs,9-3747
Langerhans cells and melanocytes of the skin,8,37 chondrocytes,46 adipocytes,28 histiocytosis X cells,37 and myoepithelial cells.34 A number of tumors of both neuronal
and nonneural origin have been reported to contain S!0011.12.19,22.34.36,38.41.44,49 ^
^
w e
h a y e
use(j S
.,
0 0
a s
a marker to study the distribution of Schwann and glial
cells in peripheral tissues.13,43
In the present study, 23 urinary bladders from multiple
sclerosis patients with various symptoms were immunostained using an antiserum to S-100, which was used
here as a marker for Schwann cells. The findings were
compared with those obtained from 136 nonmultiple
sclerosis bladders with dysfunctional symptoms and
those from normal controls.
Materials and Methods
Specimens of dysfunctional bladders from multiple
sclerosis patients (n = 23) were taken from the dome
and the trigone of the bladder by endoscopic or surgical
biopsy. The endoscopical biopsies were small pieces
(about 0.4 cm3) and consisted of the transitional epithelium and about one third of the muscle layer. Surgical
samples were larger (about 1.5 cm3) and consisted of
the whole thickness of the bladder wall. Control tissues
were taken from dysfunctional bladders of nonmultiple
sclerosis patients (n = 136), including those with primary
or secondary bladder instability, acontractile bladder,
and sphincter weakness, and from normal urinary bladders (n = 21). The causes for the bladder dysfunction
in the nonmultiple sclerosis group included bladder neck
obstruction, cystitis, trauma, spinal compression, spina
bifida, prostatic carcinoma, bladder carcinoma, nerve
damage at operation, and idiopathic bladder instability.
The symptoms of the patients with dysfunctional bladders
650
A.J.C.P. • December 1984
GU ET AL.
due to multiple sclerosis or other causes included urge
incontinence, stress incontinence, enuresis, and urine
retention. The "normality" of the urinary bladder was
established by lack of symptoms, a normal voided
volume chart, a normal flow rate, and absence of
residual urine. The normal bladder tissues were biopsies
taken at follow-up investigation after resection of transitional epithelial carcinomas or prostatic carcinomas.
A minor proportion of the control tissues in this study
have been used in a separate study to investigate peptidecontaining nerves in unstable bladder.17
The tissues were fixed, immediately after removal, in
0.4% benzoquinone in phosphate-buffered saline (PBS)
for one to two hours, according to the size of the tissue,
at room temperature.' After washing in PBS containing
7% sucrose at 4 °C overnight, the tissues were frozen
and sectioned at a thickness of 10 nm in a cryostat at
—20 °C. The sections were mounted on poly-L-lysine
(PLL)-coated glass slides21 and left to dry at room
temperature for 40 minutes. A modified procedure of
the indirect immunofluorescence method 16 was carried
out using an antiserum to S-100. The dilution for the
first layer antiserum was 1:800 in PBS. Routine controls
were carried out, including preabsorption of the antiserum with S-100 antigen, using normal rabbit serum as
the first layer and omission of the first layer. The
antiserum was raised in New Zealand white rabbit
against natural bovine S-100 and characterized according
to Zuckerman and associates.50 One section from each
block also was stained with hematoxylin and eosin. The
sections were examined and graded by two persons
independently, without knowing the diagnosis for each
case.
Results
No apparent changes were observed in the hematoxylin
and eosin preparations (Fig. 1). An infiltration by lymphocytes, polymorphonuclear cells, and macrophages
was seen in cystitis and, occasinally, in other types of
dysfunctional bladder, indicating acute or chronic local
inflammation. In the bladders from multiple sclerosis
patients, however, a slight or moderate cellular infiltration
was evident in only 5 out of the 23 cases (Fig. 2).
S-100 immunoreactivity was found to be present in
most of the tissues studied. The immunoreactivity was
distributed in elongated fibers, or spots when cut transversely, in all the histologic layers of the bladder. This
appearance was in keeping with the location of S-100
in Schwann cells. There were significant increases in S100 immunoreactivity in almost all the bladders from
patients with multiple sclerosis as compared with those
from patients with other bladder dysfunctions and from
those with normal bladders. The extent of this increase
varied in different cases. In some patients, at least a
threefold to fivefold increase in the abundance of S-100
immunoreactivity was observed. The increased S-100
immunoreactivity in the multiple sclerosis bladder was
particularly concentrated in the smooth bundles, where
networks of fibers with swellings containing unstained
nuclei were formed, strongly suggestive of Schwann cell
hyperplasia (Figs. 3 and 4). The amount of S-100
immunoreactivity in the normal bladders was quite
consistent, being apparently lower than that in the
multiple sclerosis patients. The amount of S-100 immunoreactivity in the dysfunctional bladders from patients without multiple sclerosis showed a larger variation
but was mostly in the normal range. In this group, the
amount of S-100 immunoreactivity apparently was increased in 26 cases, particularly in those of cystitis, and
was absent in 6 cases, in whom the bladders were
paralyzed and distended. In contrast, the increase in S100 immunoreactivity occurred in most of the bladders
from multiple sclerosis patients, of which only five
showed local inflammation. In one multiple sclerosis
patient, in whom the bladder was distended and nonfunctional, the S-100 immunoreactivity was absent. The
results are shown in Figure 5. All the controls for
immunocytochemistry were negative.
Discussion
In the present study, it has been found that the
abundance of S-100 immunoreactivity was increased
markedly in the urinary bladders from multiple sclerosis
patients as compared with the control tissues. S-100 has
been reported to occur in a number of cellular components. 5-8.26-28,31,34,37,46,47 However, in the urinary bladder
these components are either absent or present in very
small quantities, except Schwann cells. In addition, the
frequency and appearance of the S-100 immunoreactivity
resembles only Schwann cells, and the increased S-100
immunoreactivity fits with Schwann cell hyperplasia.
Therefore, it appears that Schwann cell proliferation
exists in the urinary bladder of multiple sclerosis patients
and this was not readily identifiable on hematoxylin and
eosin preparations.
Numerous investigations have been carried out to
study the cause and the pathogenesis of multiple sclerosis,
and some have sought to elucidate why the lesions
occurred exclusively in the central nervous system.10,25
It has been postulated that certain unsuppressed immunoreactions occurred between antibody and brain
tissue antigen (mainly myelin), which might be caused
by a number of factors. However, none of the theories
are conclusive, and there are many controversial opinions
on the pathogenesis of this disease.
Nevertheless, much evidence has been presented that
| %*r
f * tf* ^ w
^? ^ ^
FIG. 1 (upper, left). A multiple sclerosis urinary bladder. Note that a moderate cellular infiltration
is present in the submucosa and detrusor muscle layer. Hematoxylin and eosin (X200).
FlG. 2 (upper, right). The detrusor muscle in a urinary bladder from a multiple sclerosis patient. Note
that no obvious abnormality was found on this preparation. Hematoxylin and eosin (X200).
FIG. 3 (lower, left). The detrusor muscle of a normal urinary bladder S-100 (X200).
FlG. 4 (lower, right). The detrusor muscle of a dysfunctional urinary bladder from a multiple sclerosis
patient. Note that the density of Schwann cells is increased markedly S-100 (X200).
652
GU ET AL.
++++
+++
++
+
Normal Bladder
(n = 21)
Dysfunctional
Bladder of Non-MS
Patients
(n = 136)
Bladder of
MS Patients
(n = 23)
FIG. 5. S-100 immunoreactivity in the bladders of multiple sclerosis
and control patients. Key: - negative; + small number of immunoreactive nerves; ++ moderate number of immunoreactive nerves;
+++ large number of immunoreactive nerves; and ++++ very
abundant immunoreactive nerves.
the lesions are likely to be caused by an autoimmune
reaction.25 If that is the case, the peripheral myelin may
be as vulnerable as the central, since no immunogen
specific to the central nervous system of multiple sclerosis
patients has been identified convincingly.
It has been shown that neuronal damage such as that
found in Wallerian degeneration of rabbit optic nerve35
and retrograde degeneration of neurons in the dorsal
thalamus4 is accompanied by an increase in the amount
of S-100. S-100 levels are elevated both in the cerebrospinal fluid and in the plasma of patients after strokes
and during the course of other neurologic and nonneurologic diseases, including multiple sclerosis.29,32
In the adult urinary bladder, Schwann cell hyperplasia
indicated by increased S-100 immunoreactivity may be
presumed to occur in two sets of circumstances, apart
from Schwannoma: one is the regeneration process,
following nerve fiber destruction; and the other is nerve
demyelination, which is the principal change in the
brain lesion of multiple sclerosis. Both these processes
indicate that there are structural lesions in the neurons
of the urinary bladder. In some cases of the present
study, this nerve damage might be caused by local
inflammation. This was supported by the finding of
marked increases in S-100 immunoreactivity in the
bladders with cystitis from patients with multiple sclerosis
and without multiple sclerosis. However, in most multiple sclerosis bladders, no signs of inflammation were
found and the Schwann cell hyperplasia was still present.
AJ.C.P. • December 1984
Therefore, it is likely that histopathologic changes in
multiple sclerosis are not restricted to the central nervous
system but also occur in the peripheral nerves. The
bladder symptoms thus may be caused directly by the
neuronal lesion in the bladder, instead of, or, in addition
to, indirectly by the lesions in the central nervous
system. It is unlikely that the change in the bladder is
secondary to the lesions in the central nervous system,
since there have been no reports that damaged central
neurons lead to a severe proliferation of Schwann cells
in the controlled organ, although transneuronal atrophic
changes may, in a few instances, occur in the secondary
ganglia and their axons.48 The possible destruction of
nerve fibers in the multiple sclerosis bladder also was
indicated by a decrease in vasoactive intestinal polypeptide- (VIP) containing nerves in most multiple sclerosis
specimens, which was found in a separate investigation
into the involvement of peptide-containing nerves in
bladder pathology (unpublished observation).
The cytochemical composition of the central and the
peripheral myelins differ significantly.15 However, one
protein common to both the central and the peripheral
myelins, the basic protein,214 has been found to be the
antigen responsible for inducing autoimmune diseases
in the brain.24 The fact that the central and the peripheral
glial elements can react with the same S-100 antibody
suggests that immunogens common to both sites exist.
Therefore, the destruction of myelin may occur in the
peripheral nerves as well as in the central nervous system
as a result of the same pathogenic causes. Pette and
collaborators39,40 observed that an autoimmune response
against peripheral myelin can be provoked in experimental animals by injection of homogenized Schwann
cell and Freund's adjuvant. This may give some indication to the possible immunopathogenic mechanism of
the peripheral lesions in multiple sclerosis patients. Pette
and colleagues found also that severe demyelination
occurred in the peripheral nerves of rabbits, 9-12 days
after innoculation with cultures of Schwann cells from
monkeys and rabbits. It is noteworthy that this demyelination took place in the absence of histiocytic and
lymphocytic infiltration of the affected nerves. An identical lack of severe cellular infiltration in the urinary
bladder of multiple sclerosis patients was found in this
study.
One of the differences between peripheral and central
myelin is that in the periphery one internodal myelin
segment is formed by one Schwann cell and its destruction is readily repaired. In contrast, in the central
nervous system, a single oligodendrocyte is connected
to many myelin segments by long cytoplasmic projections.320 The damage to one oligodendrocyte could
cause the disintegration of many myelin segments and
result in more widespread and lasting demyelination,
Vol. 82 • No. 6
S-100 IN MULTIPLE SCLEROSIS BLADDER
23
which is far more difficult to repair. This might be one
of the reasons why the peripheral lesions in multiple
sclerosis patients are minor in comparison with the
central damage and have been neglected.
Another important fact is that the urinary bladder
often is one of the earliest organs in the periphery to
develop symptoms in a multiple sclerosis patient. The
bladder involvement is so common that Prineas estimated that it existed in 78% of multiple sclerosis patients.42 It is not surprising to find that the bladder
dysfunction often is correlated closely with the severity
of the disease,30 since the pathogenesis in the periphery
and in the central nervous system may be more or less
the same. Presumably, a mild attack would demyelinate
and damage nerves to a certain extent and cause bladder
instability resulting in enuresis and urge incontinence.
A severe attack would destroy more nerves and cause
cessation of the neural response in the bladder, resulting
in an extended bladder and urine retention.
The present finding provides evidence to show that
primary peripheral neuronal lesions may exist in multiple
sclerosis. Therefore, we feel that the concept of multiple
sclerosis being a disease wholly of the central nervous
system should be reexamined.
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