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Increases levels of circulating intercellular adhesion molecule-1 in multiple sclerosis and human T-lymphotropic virus type I-associated myelopathy.

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BRIEF COMMUNICATIONS
Increased Levels of
Circulating Intercellular
Adhesion-Molecule- 1 in
Multiple Sclerosis and
Human T-1vmDhotrooic
Virus Type' I-AssocGted
Myelopathy
N. Tsukada, MD, PhD, K. Miyagi, MD, PhD,
M. Matsuda, MD, PhD, and N. Yanagisawa, MD, PhD
We evaluated the presence of soluble intercellular adhesion molecule-1 (SICAM-I)antigen in the sera of patients
with multiple sclerosis and human T-lymphotropic virus
type I (HTLV-I)-associated myelopathy (HAM) using
an enzyme-linked immunosorbent assay. Patients with
multiple sclerosis in the active phase had higher sICAM
serum levels than did control subjects ( p < 0.01). In
addition, a significantly increased serum level of
sICAM-I was found in patients with HAM ( p < 0.001).
Furthermore, we found a positive correlation with HAM
sICAM-1 and tumor necrosis factor-a levels in the sera
of patients with multiple sclerosis in the active phase (Y
= 0.88, p < 0.01) and in those with HAM (Y = 0.86, p
< 0.01). These results suggest that serum SICAM-1may
be related to clinical activity in patients with multiple
sclerosis and the detection of sICAM-1 could be useful
as a marker of inflammatory disease.
Tsukada N, Miyagi K, Matsuda M, Yanagisawa N.
Increased levels of intercellular adhesion
molecule- 1 in multiple sclerosis and human
T-lymphotropic virus type I-associated
myelopathy. Ann Neurol 1993;33:646-649
The damage to the blood-brain barrier in multiple sclerosis (MS) is probably due to an immunological mechanism 11, 21. It has been suggested that the adhesion
of lymphocytes to cerebral endothelial cells plays an
important role in the early stages of the immune response, which is characterized by infiltration of mononuclear cells into the central nervous system (CNS)
133. Although lymphocytes or leukocytes attached to
the cerebral endothelium have been identified in immune disorders associated with CNS inflammation, inFrom the Department of Neurology, Shinshu University, School of
Medicine, Matsumoro, Japan
Received Oct 13, 1992, and in revised form Dec 15 Accepted for
publication Jan 15, 1993.
Address correspondence to Dr Tsukada, Department of Health
Medical Center, Shinshu University, Asahi 3-1-1, Matsumoto (390),
Japan.
cluding MS, the mechanisms by which these cells migrate across the blood-brain barrier into the CNS is
unknown. Interaction between intercellular adhesion
molecule-1 (ICAM-l), a member of the immunoglobulin supergene family, and lymphocyte functionassociated antigen-1 (LFA-l), a member of the integrin
family, may facilitate the migration of lymphocytes into
the CNS. ICAM-1 is induced on epithelial cells, fibroblasts, and endothelial cells by cytokines secreted
by mononuclear cells, including tumor necrosis factor
(TNF), interleukin-1 (IL-l), and interferon-? (IFN-y)
143. Recently, Marlin and colleagues 151 purified the
soluble form of ICAM-1 (SICAM-1) which is normally
membrane-bound. A circulating form of sICAM-1 has
also been identified in normal human serum using enzyme-linked immunosorbent assay (ELISA). Serum
levels of cytokines, such as IFN-y, IL-1, and TNF-a,
are increased in patients with MS, particularly during
exacerbations, suggesting that cytokines may contribute to acute relapses of MS C6-81. Recently, Sobel and
coauthors [9] reported that numerous ICAM- 1positive vessels were present in plaque edges from patients with acute MS.
Human T-lymphotropic virus type I (HTLV-I)-associated neurological disease mainly involving the spinal cord has been designated as HTLV-I-associated
myelopathy (HAM)/tropical spastic paraparesis (TSP)
1103. Pathological evidence suggests that the bloodbrain barrier is involved, but a direct causal relationship
between virus infection and the damage to the bloodbrain barrier has not been demonstrated. Expression
of ICAM-1 on mononuclear and cerebral endothelial
and glial cells in sites of active inflammation further
implicates interactions between LFA-1 and ICAM-1 in
the immune response. We measured serum levels of
sICAM-1 and TNF-a in patients with MS and HAM
using ELISA, for the purpose of determining whether
increased levels of ICAM-1 are related to the severity
of blood-brain barrier damage in MS and HAM.
Materials and Methods
The study population consisted of 31 patients with MS, 13
with HAM, 10 non-HAM carriers, 15 patients with other
neurological diseases, and 15 normal control subjects.
Patients with Multiple Sclerosis
We clinically diagnosed 3 1 patients as having definite MS
according to the criteria of Poser and colleagues 11I]. There
were 11 males and 20 females, and the age range was 15 to
59 years (mean age, 38 years). Patients were classified as
having either acute relapsing MS (25 patients; mean number
of exacerbations, 2.5Jyear) or chronic progressive MS (6 patients). Patients with acute relapsing MS were divided into
two groups: those in the active phase (clinical exacerbation),
14 patients (mean duration of disease, 3.5 years); and those
in the inactive phase (clinical remission), 11 patients (mean
duration of disease, 5.6 years). Blood samples were taken
646 Copyright 0 1993 by the American Neurological Association
either during an exacerbation or remission. The clinical diagnosis of MS was based on clinical signs supported by obiective findings. The latter included the presence of lesions confirmed by magnetic resonance imaging (MRI) or computed
tomography (CT), the finding of oligoclonal bands, and increased IgG levels in the cerebrospinal fluid. Blood samples
were taken within 3 days after an exacerbation of the disease.
The patients represented those with typical MS found worldwide, not only those whose MS involved the spinal cord
exclusively. Patients with acute MS who are Japanese often
manifest the latter syndrome. N o anti-HTLV-I antibody was
found in the sera of patients with MS. MS patients with
intercurrent infections were excluded from the study.
In 7 of 14 patients with acute relapsing MS, serum
ICAM-1 levels were measured twice, during an exacerbation
and in remission after treatment by conicosteroids. Other
samples were obtained prior to treatment with corticosteroids, immunosuppressive agents, or the use of plasmapheresis.
HAM and non-HAM Carriers
HAM was diagnosed in 13 patients (7 men and 6 women;
age range, 35-70 years; mean age, 58.1 years) according to
the criteria of Osame arid colleagues [lo]. HTLV-I antibody
titers, measured by indirect immunofluorescence, were
above 80 IF in patients with HAM. HTLV-I antibody titers
in 10 HTLV-I-seropositive patients ( 5 men and 5 women;
age range, 33-72 years) who did not have HAM were above
40 IF. Blood samples were also obtained before treatment
with corticosteroids.
Normal Control Szlbjects
Serum samples from 15
' healthy individuals (6 men and 9
women; age range, 27-62 years; mean age, 40.5 years) were
examined. Control subjects with infection were also excluded
in this study.
All serum samples were quickly frozen and stored at
- 70°C until the time of the assay.
Assay of ICAM-1
Serum ICAM- 1 levels were measured by double-determinant immunoassay using a commercially available ELISA kit
(Bender, Vienna, Austria). Briefly, serum samples diluted
1:100 with buffered protein matrix were added to microwell
strips coated with a murine monoclonal antibody to human
SICAM-1. After being washed twice with a buffer (Tween
20), the microwell strips were incubated with horseradish
peroxidase-con jugated anti-SIC AM- 1 murine monoclonal
antibody diluted 1:100 with the assay buffer. The reaction
was stopped after 15 minutes by the addition of 4N sulfuric
acid. The absorbance of each well was measured with a Beckman (Fullerton, CA) LS-5800 spectrophotometer. A standard
curve was used to determine the sICAM-1 concentration in
each sample. The detection limit for ICAM-1 was 0.625 ng/
ml.
Tumor Necrosis Factor-a Assay
We previously developed a specific and sensitive sandwich
enzyme immunoassay for human TNF-a in serum 1121.
Briefly, a monoclonal anti-human TNF-a IgGl (E43)-
coated polystyrene ball was incubated with recombinant human TNF-a or test serum. After the reaction mixture was
removed, the polystyrene ball was washed twice and then
incubated with anti-human TNF-a Fab' (F12)-peroxidase
conjugate. Peroxidase activity bound to the polystyrene ball
was assayed at 30°C for 1 hour using 3-(4-hydroxyphenyl)propionic acid as a substrate. Fluorescent intensity was measured by a Hitachi fluorescence spectrophotometer (F-40 10,
Tokyo, Japan). The detection limit for human TNF-a was
3.4 ngiliter in 5.0 ~1 of serum.
statistical Analysis
Statistical analyses were performed using Student's t test and
paired t test. The correlation coefficient r test was also used
to compare SICAM-1 to TNF-a levels.
Results
Soluble ICAM-I LweIs in Multiple Sclerosis and
HAM Patients
Patients with acute relapsing MS in the acute phase
(299.2 2 7 1.1 ngiml) had significantly increased levels
of SICAM-1 compared with those in the inactive phase
(187.0 ? 33.4 ng/ml, p < 0.01) and control subjects
(173.6 k 56.3 ng/ml, p < 0.01) (Fig 1). T h e r e was
no significant difference in sICAM- 1 levels between
patients with MS in the inactive phase and control subjects. Steroids lowered the serum ICAM-1 levels of 7
patients with acute relapsing MS during an exacerbation.
Fig I. Levels of soluble intercellular adhesion molecule-1
(SICAM-1) in the sera of patients with multiple sclerosis (MS)
and human T-lymphotropicvirus type I-associated myelopatby
(HAM). slCAM-1 serwn levels in patients with MS and
HAM were significantly increased as compared with control levels (p < 0.01 and p < 0.001, respectivelyl. ControlsICAM-1
levels are shown in the last column. Values are expressed as
means standard deviations.
*
p<o.001
p4.01
450
7 300
a0
44
!
+i
200
I00
I
active
inactive
HAM Non-HAM control
carrier
(n-14)
(n=11)
(n=13)
acute
relapsing
type
(-25)
@=lo)
(n=15)
chronic
progressive
type
(d)
MS
Brief Communication: Tsukada et al: Increased ICAM-1 in MS and HAM 647
0
20 -
f
ooo/
0
e
lL
15-
c
0
-a
i?
Y)
/.
10-
f
2
%
100
200
-
0 .
300
400
500 nglml
Serum levels of slCAM-1 antigen
Acute relapsing MS (exacerbation )
o HAM
Fig 2. Correlation between levels of soluble intercellular adhesion
molecule-1 (SICAM-1) and tumor necrosis factor-a (TNF-a) in
the sera of patients with acute relapsing multiple sclerosis
(closed circles) during an exacerbation (n = 14, r = 0.88,
p < 0.01) andpatients with human T-lymphotropzc virus type
I-associated myelopathy (open circles) (n = 10, r = 0.86,
p < 0.01).
Patients with HAM (340.7 ?c 72.9 ng/ml) had significantly higher levels of SICAM-1 compared with
HTLV-1-seropositive non-HAM carriers (168.0 &
57.4 ng/ml, p < 0.001) and control subjects ( p <
0.001). There were no significant differences in
SICAM-1 levels between HTLV-I-seropositive nonHAM carriers and control subjects.
We measured sICAM-1 levels in control patients
twice in 1 month to examine the normal variability of
ICAM-1. Levels of SICAM-1 constantly ranged from
82 to 300 ng/ml in sera of control subjects (first measurement, 173.6 f. 56.3 ng/ml; second measurement,
159.5 k 48.1 nglml).
Serum ICAM-1 and Tumor Necrosis Factor-a Levels
There was a positive correlation between SICAM-1 and
TNF-a levels in patients with acute relapsing MS in
the active phase, as determined by the correlation coefficient Y test (Y = 0.88, p < 0.01) (Fig 2). A positive
correlation was also found between sICAM-1 and
TNF-a levels in patients with HAM ( Y = 0.86, p <
0.01).
Discussion
We investigated serum levels of sICAM-1 in MS,
an autoimmune demyelinating disease-associated inflammation, during an exacerbation. Serum levels of
SICAM-1 antigen were also measured in patients with
648 Annals of Neurology Vol 33 No 6 June 1993
HAM, which is characterized histologically by gliosis
and mononuclear cell infiltrates around blood vessels.
ICAM-1 expression by cytokines appears to be involved in the regulation of leukocyte localization in
inflammatory diseases 1131. It has been reported that
ICAM-1 is expressed on the surface of resting peripheral blood leukocytes and that its expression is increased by the activation of monocytes and T and B
lymphocytes 1141. The origin of sICAM-1 can be diverse and may be from mononuclear cells or endothelid cells. Expression of ICAM-1 in the normal CNS,
like that on human monocytes, might vary among individuals, but this study also supported the previous report that levels of SICAM-1 ranged between 100 and
200 ng/ml in sera from normal individuals 1157. Vascular ICAM-1 may, therefore, be involved in regulation
of the focal accumulation and infiltration of leukocytes
in the CNS immune reaction. TNF-a, a potent lymphokine, can induce expression of ICAM-1 on endothelial cells. ICAM-1 expression on cerebral endothelid cells has been detected in brain samples from
patients with active MS, using immunoperoxidase
staining for ICAM-1 191. In MS, where large numbers
of lymphocytes enter the CNS and contribute to the
inflammatory response, dense ICAM- 1 expression on
endothelium may be responsible for some aspect of
perivascular infiltration as well as the antigen-presentation reaction.
We observed a significant correlation between
SICAM-1 and TNF-a levels in patients with acute relapsing MS in the active phase. Increased SICAM-1
levels may reflect activation of endothelial cells in such
patients and may be related to clinical activity.
Patients with HAM also exhibited increased
sICAM- 1 levels. Perivascular infiltration is common to
HAM and MS, and the size and location of affected
vessels are similar in both diseases. Our previous data
1161 showed that soluble CD8 and soluble IL-2 receptor (sIL-2R) were increased in the sera of patients with
HAM. Thus, these observations also support the proposal that there are increased activated T cells in patients with HAM C171. However, the direct relationship between HAM and HTLV-I remains unknown.
The increased SICAM-1 levels in patients with acute
relapsing MS suggest that ICAM-1 may contribute to
lymphocyte migration into areas of inflammation in the
CNS. It was recently reported that serum levels of
cytokines such as TNF-a and IL-1 are increased in
patients with acute relapsing MS during an exacerbation 161. These cytokines induce adhesion of endothelial cells to lymphocytes and play an important role in
the exacerbation of vascular damage in MS. An increase in sIL-2R levels in serum was also found in patients with acute MS in the active phase 1167. TNF-a
has been histochemically identified in active lesions in
MS, and a previous report 181 indicated that TNF-a
plays a major role in blood-brain barrier permeability
during an exacerbation of MS. It will be worthwhile to
determine if the reciprocal changes in serum levels of
sICAM-1 from patients with MS and HAM are related
to the pathogenesis of vascular lesions in these diseases.
The present data suggest that quantitative assessment of sICAM-I may be of value in detecting or
evaluating inflammatory diseases.
15. Rothlein R, Mainolfi EA, Czajkowski M , Marlin SD. A form of
circulating ICAM-1 in human serum. J Immunol 1991;147:
3788-3793
16. Tsukada N, Matsuda M, Miyagi K, Yanagisawa N. Soluble CD4
and CD8 in the peripheral blood of patients with multiple sclerosis and HTLV-I-associated myeloparhy. J Neuroimmunol
1991;35:285-293
17. Itoyama Y, Kira J, Fujii N, Goto I, Yamamoto N. Increases in
helper/inducer T cells and activated T cells in HTLV-I associated myelopathy. Ann Neurol 1989;26:257-262
This work was supported by grants from the Intractable Disease
Division, Public Health and Welfare of Japan.
Coexistence of Hereditary
Motor and Sensory
Neuropathy Type Ia and
IgM Paraproteinemic
Neuropathy
The authors would like to thank D r M. Osame (Department of
Neurology, Kagoshima University) and Dr. Y. Itoyama (Department
of Neurology, Kyushu University) for collaborative work, and Miss
A. Kiyosawa for technical assistance.
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myelopathy associated with elevated antibodies to human Tlymphotropic virus type I and adult T-cell leukemia like cells.
Ann Neurol 1987;2 1:117-122
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necrosis factor-a. Clin Chem Entym Comms 1990;3:1-8
13 Dustin ML, Staunton DE, Springer TA. Supergene families
meet in the immune system. Immunol Today 1988;9:213-215
14 Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA.
Induction by IL-1 and interferon-y: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-I).
J Immunol 1986;137:245-254
R. Gregory, MRCP," P. K. Thomas, DSc,"?
R. H. M. King, PhD,t P. L. J. Hallam, BScJ
S. Malcolm, PhD,$ R. A. C. Hughes, FRCP,$ and
A. E. Harding, FRCP"
A patient with minimal motor dysfunction dating from
early childhood developed more rapidly progressive distal weakness and positive sensory symptoms due to peripheral neuropathy in the fourth decade of life. DNA
analysis showed the partial duplication of chromosome
17p associated with hereditary motor and sensory neuropathy type Ia. In addition, the patient had an IgM
paraproteinemia and the typical morphological features
of IgM paraproteinemic neuropathy on nerve biospsy.
Gregory R, Thomas PK, King RHM, Hallam PLJ,
Malcolm S, Hughes RAC, Harding AE.
Coexistence of hereditary motor and sensory
neuropathy type Ia and IgM paraproteinemic
neuropathy. Ann Neurol 1993;33:649-652
Charcot-Marie-Tooth disease is clinically and genetically heterogeneous 111. The most common of these
disorders is type Ia hereditary motor and sensory neuropathy (HMSN), an autosomal dominant demyelinating neuropathy that maps to chromosome 17 and is
associated with a duplication of band p 11.2 containing
From the "University Department of Clinical Neurology, Institute
of Neurology, tDepartment of Neurological Science, Royal Free
Hospital School of Medicine, $Molecular Genetics Unit, Institute
of Child Health, and $Department of Neurology, UMDS, Guy's
Hospital, London, UK.
Received Oct 13, 1992, and in revised form Jan 27, 1993. Accepted
for publication Jan 28, 1993.
Address correspondence to Prof Harding, University Department of
Clinical Neurology, Institute of Neurology, Queen Square, London,
W C l N 3BG. UK.
Copyright 0 1993 by the American Neurological Association 649
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associates, level, molecules, virus, typed, human, myelopathy, circulating, lymphotropic, increase, sclerosis, multiple, intercellular, adhesion
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