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Circulating adhesion molecules and tumor necrosis factor receptor in multiple sclerosis Correlation with magnetic resonance imaging.

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Circulating Adhesion Molecules and Tumor
-
Resonance Imagng
Hans-Peter Hartung, MD," Karlheinz Reiners, MD," Juan J. Archelos, MD," Marco Michels, MD,'
Pierrette Seeldrayers, MD,"t Fedor Heidenreich, MDJB Karl W. Pflughaupt, PhD," and Klaus V. Toyka, MD"
~~
Adhesion molecules are important in T-cell trafficking to sites of inflammation. We determined levels of circulating
vascular cell adhesion molecule-l(VCAM- l), Lselectin, and E-selectin in the serum of 147 patients with definite
multiple sclerosis of the remitting-relapsing or secondary progressive type. Soluble VCAM-1 and L-selectin concentrations were increased compared to levels in a large group of control subjects. Levels were highest in patients with
gadolinium-enhancing lesions on magnetic resonance imaging (VCAM-1: 1,011 k 276 vs 626 f 87 ng/ml; L-selectin:
1,130 f 272 vs 793
207 ng/ml [mean f standard deviation}; p < 0.0001 vs patients without enhancing lesions).
Serum levels of soluble tumor necrosis factor receptor (60kd) were also raised (2.64 2 1.23 vs 2.17 2 0.69 ng/ml in
subjects with other neurological diseases and 2.1 f 0.77 n g / d in healthy control subjects; p < 0.05). Soluble VCAM-I
and Gselectin levels were correlated to concentrations of soluble tumor necrosis factor receptor. In 13 patients with
viral encephalitis, similar observations were made. Raised levels of soluble VCAM-1 and L-selectin probably reflect
cytokine-induced endothelial cell and T-lymphocytelmonocyte activation occurring in the process of T-cell migration
into the central nervous system. Tumor necrosis factor-alpha may be critically involved.
*
Hartung H-P, Reiners K, Archelos JJ, Michels M, Seeldrayers P, Heidenreich F, Pflughaupt KW, Toyka KV.
Circulating adhesion molecules and tumor necrosis factor receptor in multiple sclerosis:
correlation with magnetic resonance imaging. A n n Neurol 1995;38:186- 193
Pathological observations, numerous immunological
studies, and findings in the animal model of experimental allergic encephalomyelitis (EAE) have firmly established the pivotal role of aberrant T-cell responses directed to myelin antigens in the pathogenesis of
multiple sclerosis (MS). Autoreactive T lymphocytes
circulate in the blood and cerebrospinal fluid (CSF) of
MS patients but also can be retrieved, possibly with
lower frequency, from normal individuals [l, 2).
Hence, additional factors must operate that render
such autoreactive T cells autoaggressive to cause inflammatory demyelination. T cells must leave the systemic circulation and access the central nervous system
(CNS) to initiate local autoimmune responses. T-cell
trafficking is governed by an ensemble of adhesion
molecules, which are reciprocally expressed on leukocytes and endothelial cells {33. An intricately coordinated sequential interaction of these molecules is required for T-cell migration across the blood-brain
barrier to occur. The mechanisms underlying this pro-
cess of homing and invasion in MS recently came under close scrutiny {4, 51. In general, the process of
extravasation of leukocytes at sites of inflammation can
be dissected into at least three distinct steps 161. Initially, leukocytes under shear stress engage in rolling
movements and weakly attach to the vessel wall. The
selectin group of adhesion molecules is of paramount
importance at this stage of tethering. Secondly, rolling
leukocytes are arrested at the vascular lining under the
influence of chemoattractants such as chemokines,
CSa, and platelet-activating factor [ 3 ] . Finally, the interaction of integrins like intercellular adhesion rnolecule- 1 (ICAM-1) and vascular cell adhesion molecule- 1
(VCAM-1) with their respective ligands promotes
strong adhesion to endothelium before transendothelial migration occurs [6]. Adhesion molecule expression is greatly upregulated by cytokines generated in
the course of an inflammatory response 13, 61. Eselectin, L-selectin, ICAM-l, and VCAM-1 are rapidly
shed from activated endothelial cells, lymphocytes, or
From the "Departments of Neurology, Julius-MaximiliansUniversitat, Wurzburg, Germany; tErasmus University Hospital,
Bruxelles, Belgium; $Heinrich Heine-Universifat, Dusseldorf; and
IMedizinische Hochschule, Hannover, Germany.
Received Nov 23, 1994, and in revised form Mar 6 and Apr 3,
1995. Accepted for publication Apr 5 , 1995.
Address correspondence to Dr Hartung, Department of Neurology,
University of Wurzburg, Josef-Schneider-Strase 11, D-97080
Wurzburg, Germany.
186 Copyright 0 1995 by the American Neurological Association
Table 1 . Adhesion Molecules: Characteristics and Ligands"
Adhesion Molecules
Size
Cellular Distribution
Ligands
ICAM-1 (CD54) (intercelMar adhesion molecule)
90-115 kd
LFA-1 (CD18/CDlla)
Mac-UCR3
(CD18/CD 1la)
VCAM-1 (CD106) (VZCUlar cell adhesion molecule)
VLA-4 (a4p 1 integrin)
CD49d/CD29) (very
late activating antigen-4)
LFA-1 (CDlldCD18)
(leukocyte functionassociated molecule-1)
L-selectin (CD62L) (leukocyte adhesion molecule- 1)
E-selectin (CD62E) (endothelial leukocyte adhesion molecule-1)
90-110 kd
B cells, T cells, endothelial
cells, monocytes/macrophages, microglia, dendritic cells, and others
Endothelial cells, macrophages, dendritic cells,
and others
T lymphocytes
180 kd (a-chain)
95 kd @-chain)
Leukocytes
74 kd (leukocytes)
90-100 kd (neutrophils)
T cells, neutrophils, monocytes, eosinophils, basophils, natural killer cells
Endothelial cells
ICAM-1
ICAM-2
ICAM-3
MAdCAM-1
CD34
GIyCAM- l
Sialylated Lewis a, x-like
carbohydrates
150 kd (a-chain)
120-130 kd @-chain)
107-115 kd
VLA-4 (a4pl integrin)
(CD49d/CD29)
a4p7 integrin
VCAM-1, fibronectin
'Compiled from Springer [3] and Bevilacqua [bJ.
monocytes (Table l),and soluble forms of these molecules can be measured in body fluids (7-11).
Recently several groups documented raised levels of
soluble ICAM-1 in the serum and CSF of MS patients.
Levels were correlated to the degree of blood-barrier
damage 112-141.
We examined sera from a large cohort of MS patients for the presence of the soluble adhesion molecules Lselectin, E-selectin, and VCAM-1 and correlated them with clinical and magnetic resonance
imaging (MRI) evidence of disease activity. We also
quantitated serum levels of the soluble 60-kd tumor
necrosis factor (TNF) receptor (sTNFR) which binds
TNF-alpha 115-171, the cytokine considered to be of
prime importance in inflammatory demyelination and
a strong inducer of adhesion molecules [4,6, 181. Furthermore, we carried out the same assays on sera from
patients with viral encephalitides, another group of T
cell-mediated inflammatory disorders of the CNS
gressive MS were considered to have active disease when the
EDSS score had increased by at least 1 point in the 6 months
before serum sampling. Accordingly, 111 patients were
judged to have active disease while 36 had stable MS. None
of the patients had fever at the time of serum sampling.
Twenty-four patients had urinalysis evidence of a low-grade
urinary tract infection. None of the patients had immunosuppressive treatment at the time of serum sampling.
MRI was performed on each patient according to a standardized protocol outlined by Miller and colleagues [22),
using a 1.5-T system. Patients were considered to have acute
lesions when areas of hyperintensity, compared with the intensity of surrounding brain parenchyma, were recorded
on a "1-weighted sequence following gadoliniumdiethylenetriamine pentaacetic acid (Gd-DTPA) (0.1 mmoll
kg of body weight) injection. Seventy-six patients showed
enhancement. MS in 75 of these patients was considered to
be clinically active while disease in 1 patient was deemed
clinically stable.
1191.
Materials and Methods
Subjects
Serum samples were collected from
14 patients with newly diagnosed generalized myasthenia gravis (MG). All patients had circulating anti-acetylcholine receptor antibodies.
One hundred forty-seven patients
had clinically or laboratory-supported definite MS according
to the criteria of Poser and associates 120). Fifty-three were
males, and 94 females. Ninety-seven had the relapsingremitting type of the disease, with at least three relapses
occurring in the preceding 24 months, one of which occurred
within 10 days prior to serum sampling. Fifty patients had
secondary progressive MS. Disease severity was scored using
Kurtzke's Expanded Disability Status Scale (EDSS) 1211 and
the scores ranged between 1 and 8 (mean 5 standard deviation [SD): 4.6 ? 1.7). Patients with secondary chronic pro-
Samples were obtained from 13 patients with viral encephalitis within 3 days of disease onset.
The diagnosis was established on the basis of history, clinical
examination, CSF findings (pleocytosis), electroencephalographic findings (diffuse or focal slowing), and MRIs in 7
patients with herpes simplex and 3 patients with measles encephalitis. The etiological diagnosis was corroborated by serology. A more than twofold increase in serum and CSF titers
suggested Epstein-Barr virus as the etiological agent in 2 patients and cytomegalovirus in l.
MYASTHENIA GRAVIS.
MULTIPLE SCLEROSIS.
VIRAL ENCEPHALITIS.
Hartung et al: Adhesion Molecules and sTNFR in MS
187
1800
-
1800-
1400
=
1200
d
1000
2000
-
70
-
1800-
0
. .
1
0
60:
-
*
c 50
e
.
c
8
0
.
I 20
400
200
-
0-
200
.
.
MS MG ENCOND HC
-
0-
10
.
.
MS MG ENCOND HC
F i g I. Concentrations of soluble(s) adhesion molecules uascular
cell adhesion molecule-1 (VCAM-l), L-selectin, and E-selectin
were determined by enzyme-linked immunosorbent assay in sera
ofpatients with MS ( n = 1471, myasthenia gratis (n = 141
(MG). viral encephalitis (n = 1 3 ) (ENC), other noninjammutory or nondeniyelinating neurological disorders (n = 48)
(OND), and healthy control subjects (n = 15) (HC).
sVCAM-1 and sL-selectin letiels are sign;f;cant(yelmated (p <
0,0001 and < 0,002) inpatients
with MS and
encephalitis (p < 0.0001) compared t o OND patients or healthy control subjects. Whisker plots s h w the loth, 25th, 50th (median). 75th, and 90th percentiles. Values abwe the 90th and
belw the 10th percentile are plotted as points. Notches indicate the 95% confidence internal around the median. (Explanations also appb to Figs 2 through 4).
OTHER NEUROLOGICAL DISEASES. Forty-eight patients had
one of the following noninfectious, nondemyelinating disorders: Alzheimer’s disease, Parkinson’s disease, multisystem
atrophy, astrocytoma, oligodendroglioma, meningioma, normal-pressure hydrocephalus, tension headache, or degenerative disk disease. MRI was performed on 39 patients, of
which 12 with tumors had gadolinium enhancing scans.
Fifteen healthy control subjects were recruited from hospital personnel.
HEALTHY CONTROL SUBJECTS.
-
0-
.
MS MG ENCOND HC
minutes at ambient temperature, the substrate reaction was
stopped by the addition of 2 N sulfuric acid. The absorbance
of the colored reaction product was recorded with an ELISA
microplate reader at 450 nm. Concentrations of circulating
adhesion molecules in serum samples were determined by
relating absorbance values from a standard curve generated
with the respective recombinant human adhesion molecules.
Limits of detection were less than 0.5 ng/ml for soluble (5)
ICAM-1, less than 0.3 ng/ml for sL-selectin, less than 1.6
ng/ml for sE-selectin, and less than 1.8 ng/ml for sVCAM-1.
Interassay coefficients of variation were less than 11%. Spike
exceeded g2% in
assays. sTNFR (60 kd) was
also measured immunoenzymometrically following the same
basic protocol as described above for adhesion molecules
[17). Assay kits were from Bender MedSystems, Vienna,
Austria (SICAM-1, sL-selectin, sE-selectin, and sTNFR) or
from R + D Systems, British Biotechnology, Abingdon,
Oxon, United Kingdom (SVCAM-1).
Statistics
Data were analyzed using StatView 4.0 software (Abacus
Concepts, Berkeley, CA). The Mann-Whitney U test was
applied to determine the significance of differences between
groups.
Results
Sample Collection
Serum samples were collected between 1989 and 1993 and
were stored in aliquots at - 70°C.
Measurement of Serum Concentrations of Circulating
Adhesion Molecules and Tumor Necrosis Factor
Receptor
Serum concentrations of circulating adhesion molecules were
measured by two-sided enzyme-linked immunosorbent
assays (ELISAs) in which two monoclonal antibodies directed
against different epitopes of the adhesion molecules studied
were used. A capturing antibody was coated onto the walls
of microwells. Serum samples were added together with a
horseradish peroxidase-conjugated monoclonal antibody to
the respective adhesion molecule. After incubation for 15
188 Annals of Neurology
Vol 38 No 2 August 1995
Circulating Adhesion Molecules
Circulating VCAM-1 levels were elevated in patients
with MS (825 5 305 ng/ml [mean
SDI). The mean
concentration was significantly increased over that of
patients with other neurological disorders (ONDs)
(596.5
153.6 ng/ml, p < 0.0001) and healthy control subjects (539.4 k 125 ng/ml, p = 0.004) (Fig
1A).
Levels of circulating L-selectin were also moderately
increased in MS patients (968 k 295 ng/ml) compared
to those of OND patients (812
163 ng/ml, p <
0.0006) and healthy control subjects (793
184 ng/
ml, p = 0.025) (Fig 1B).
The mean concentration of circulating E-selectin was
*
*
+_
*
Table 2. Serum Lniels of Adhesion AIoleru/e.c in MS: Comparison Between Relapsiilg-Renlittitlg (RR)
and Chronic Progressioe (CP, Disease"
MSIRR
833 (633; 1,045)
sVCAM-1 (ng/ml)
sL-selectin (ng/ml) 1,010 (830; 1,299)
sE-selectin (ng/rnl) 21.0 (15.8; 29.0)
p Value
MSICP
686 (468; 910)
CO.01
<0.0001
769 (669; 905)
21.0 (15.0; 27.0) 0.69 (NS)
MSIActive
MS/Stable
877 (672; 1,099) 598 (450; 716)
795 (798; 1,207) 768 (693; 752)
21.0 (16.0; 29.0) 19.0 (11.0; 29.5)
p Value
<0.0001
<0.0001
0.33 (NS)
aLevels of the soluble(s) adhesion molecules vascular cell adhesion molecule-1 (VCAM-l), L-selectin, and E-selectin were quantitated in serum
from patients with relapsing-remitting (MYRR), progressive (MS/CP), clinically active, and clinically stable MS by enzyme-linked immunosorbent assays. Values represent median (25th; 75th percentiles).
NS
=
not significant.
vs 792 ? 254 ng/ml Cp < 0.00011). Serum levels of
circulating adhesion molecules in patients whose disease followed a relapsing-remitting course markedly
differed, depending on the presence or absence of gadolinium enhancement on MRIs. Respective values
were as follows: sVCAM-1-1,005 2 283 ng/ml (with
enhancement) and 644
177 ng/ml (without en278
hancement) ( p < 0.0001); sL-selectin-1,168
ng/ml (with enhancement) and 857 2 229 ng/ml
(without enhancement) ( p < 0.0001); and sEselectin-26 2 10 ng/ml (with enhancement) and 20
11 ng/ml (without enhancement) ( p < 0.05).
Similarly, patients with the secondary progressive
type of MS had higher mean serum levels of soluble
adhesion molecules VCAM-1 and L-selectin when
their MRIs showed gadolinium enhancement:
t 248 vs 609 t 197 ng/ml ( p <
sVCAM-1-1,036
0.0001); and sL-selectin-961 ? 162 vs 732 5 163
nglml ( p = 0.0002). By contrast, sE-selectin levels did
7 vs 21
9 ng/ml; p = 0.72).
not differ (22
slightly higher in the total group of MS patients (22.9
-+: 10.5 ng/ml) than in patients with ONDs (19.3
7.4 ng/ml, p = 0.03) but not significantly different
from that for healthy control subjects (21.9 2 8.1 ng/
ml, p > 0.1) (Fig 1C).
*
*
Circulating Adhesion Molecules in MS Patients
Grouped According to Disease Type and Activity
When the results were broken down according to the
type of MS, it was found that serum concentrations of
sVCAM-1 and sL-selectin were higher in patients with
the relapsing-remitting compared to those with the
progressive form of the disease (Table 2). When patients were grouped according to clinical disease activity, concentrations of circulating adhesion molecules
were significantly increased in those with active versus
those with stable MS (see Table 2).
Fifty (45%) of 111 patients with active disease had
sVCAM-1 levels in excess of the mean plus 2 SDs for
OND patients and healthy control subjects. Thirty-five
(32%) and 31 (28%) of 111 had L-selectin levels, and
only 14 (8%) and 9 (13%) patients had sE-selectin
levels in that range. In serum of patients with an autoimmune disorder of neuromuscular transmission, MG,
no such increases were noted (see Fig 1).
Serum concentrations of circulating adhesion molecules did not differ in patients with and those without
urinary tract infection nor was there any correlation to
serum C-reactive protein levels (not shown).
*
*
*
*
*
*
*
Correlation with Disability
Finally, we examined a possible relationship of serum
levels of circulating adhesion molecules and disability
as measured on the EDSS. A significant correlation was
only found between the concentration of sL-selectin
and EDSS score ( r = 0.269, p < 0.001).
Correlation with Magnetic Resonance
Imaging Findings
These differences became even more pronounced
when we compared patients with (n = 75) and those
without (n = 36) MRI evidence of blood-brain barrier
disturbance at the time of serum sampling (sVCAM-1:
1,011 2 276 vs 626
187 ng/ml; sL-selectin: 1,130
272 vs 793 2 207 nglrd; sE-selectin: 24
9.9 vs
20.9
9.8 ng/ml [ p = 0.041) (Fig 2). Very similar
results were obtained when we compared clinically active patients with and those without gadolinium enhancement on MRIs (sVCAM-1: 1,014 275 vs 634
191 ng/ml Cp < 0.OOOlJ; sL-selectin: 1,127 2 272
*
*
Circulating Adhesion Molecules in Viral Encephalitis
Substantial increases in the levels of circulating adhesion molecules were also noted in patients with acute
viral encephalitis. The mean concentration for
sVCAM-1 was 799 f 194 (vs 597 2 154 ng/ml for
OND patients, p < 0.0001); for sL-selectin, 1,194 2
279 ng/ml ( p < 0.0001); and for sE-selectin, 26.5 t
5.3 ng/ml ( p < 0.0001) (see Fig 1).
Circulating Soluble Tumor Necrosis Factor Receptor
in MS and Viral Encephalitis
Assaying patients' sera for sTNFR (60 kd) revealed
raised levels in MS patients (2.64 t 1.23 nglml) when
compared to M G patients (1.94 2 0.42 ng/ml), OND
Hartung et al: Adhesion Molecules and sTNFR in MS
189
1600
1400
=
E 1200
1 :
:
600
0
60
1400
.g
-
50:
1200
0
8
v)
600
3
0
0
CI
2
0
800
0
Icl
E
1000
0
Q)
lo
P
.
m
+.
40.-
Y
r
%
=
p
m
E, 1000
800
1800
1
E
\
Aa
1
2000 1
1
1800 I
30:
B
W
20-
400
2w
10
-
1
4
0
MR+ MR- OND
-
MR+ MR- OND
Fig 2. Data on all MS patients were categorized according to
the presence or absence of acute blood-brain barrier dysfunction
ar evidenced by the pmence of gadolinium-enhancing lesions on
TI-weighted magnetic resonance (MR) scans. M R = patients with enhancing lesions; M R - = patients without enhancing lesions.
+
patients (2.17 ? 0.69 ng/ml, p < 0.05), and healthy
control subjects (2.1 k 0.77 ng/ml, p < 0.05) (Fig
3A). Thirty-eight (26%) of 147 and 36 (25%) of 147
MS patients had sTNFR concentrations higher than
the mean plus 2 SDs for O N D patients and healthy
control subjects, respectively. The mean concentration
in patients with the relapsing-remitting form of the disease was higher (2.76 k 1.3 ng/ml) than that in patients with chronic progressive disease (2.25 ? 0.99
n g / d [Fig 3B)). Patients deemed to be in an active
phase of disease had a higher mean level (2.8 ? 1.3
n g / d ) than those considered to be in remission (1.96
? 0.6 ng/ml [Fig 3C)). Lkewise, patients with MRI
evidence of acute blood-brain barrier breakdown had
a mean sTNFR level greatly exceeding that for those
without such evidence (3.3 ? 1.2 ng/ml vs 1.5 ? 0.7
ng/ml {Fig 3D)). Sera from patients with clinical and
MRI activity (gadolinium enhancement) contained
higher mean concentration (3.3 & 1.2 ng/ml) than did
those from patients with clinical but no MRI activity
(2 2 0.9 ng/ml, p < 0.0001).
In patients with viral encephalitis, raised levels of
sTNFR (2.92 -+: 0.7 ng/ml; p < 0.001 vs O N D and
healthy control levels) were detected in sera (see Fig
3A).
Finally, there was no positive correlation of sTNFR
levels with disability as reflected by EDSS scores ( r =
0.096, p = 0.25). sTNFR levels did correlate with
circulating levels of all adhesion molecules examined
190 Annals of Neurology Vol 38 No 2 August 1995
0 )
0
MR+
MR- OND
(SVCAM-1: r = 0.358, p < 0.0001; SICAM-1: Y =
0.277, p < 0.04; sL-selectin: r = 0.466, p < 0.0001;
sE-selectin: r = 0.244, p < 0.05) (Fig 4).
Discussion
The principal finding of our study is the demonstration
that the adhesion molecules VCAM-1 and L-selectin
circulate in markedly increased amounts in the blood
of MS patients when they are in an active phase of
their disease. In contrast, sE-selectin levels were not
increased above those detectable in healthy control
subjects. This may result from differential regulation
of the gene expression for E-selectin and the other
adhesion molecules or may mirror the different kinetics of their shedding from cell surfaces [b]. Importantly, the rise in circulating levels of these adhesion
molecules was highest in patients who had MRI evidence of active blood-brain barrier disturbance reflected by the passage of contrast medium into the
brain parenchyma [23, 241. The most likely cellular
source of L-selectin in this setting is the T cell, whereas
VCAM-1 is probably derived from vascular endothelium. Lselectin takes part in the earliest step of endothelial adhesion of T lymphocytes by promoting rolling
and early attachment. These are transient and potentially reversible interactions that permit T cells to sample the local vascular bed for the presence of specific
trigger factors capable of activating leukocyte integrins.
Selectin-mediated binding is strengthened by the interaction of VCAM-1 displaying endothelial cells and its
counterpart very late activating antigen-4 (VLA-4) on
T cells 13, 61. In EAE in mice, VCAM-1 and L-selectin
were found to be upregulated during the induction
phase of the disease {25, 261. Cell surface expression
of VLA4 integrin on myelin basic protein-specific T-
r ,
r ,
iJ
m
4-
4-
4-
a
5-
5-
a
0
2-
2-
2-
1-
1-
8
,
1
0-
MS MG ENCOND HC
RR
0
CP
Fig 3. Soluble tumor necrosis factor receptor (sTNFR) (60 kd)
levels in MS and viral encephalitis. Sera of indicated patient
groups @or abbreviations,see legend to Fig 1 ) were assayed for
sTNFR. Levels in both MS and viral encephalitis patients were
increased over those for OND patients and healthy control subjects (p < 0.05/0.0001)(A).In MS patients, sTNFR levels
2000
2000
1750
1750
250
0
3-
3-
1a
a
3-
1
2
3
4
5
6
7
0
ACTIVE STABLE
MR-
MR+
were higher in those with relapsing-remitting (RR) than in
those with chronic progressizme (CP) disease (p < 0.0021 (B), in
those with clinically active than in those with stable disease (p
< 0.0001) (C), and in patients with gadolinium enhancing le) rejecting acute blood-brain bawier disturbance
sions (MR -Ithan in those without enhancement (MR-) (p < 0.0001)( D ) .
600
.
.*
.
*
2
3
4
1
1
0
1
sTNFR [ng/ml]
5
sTNFR [ng/ml]
6
7
0
1
2
3
4
5
6
7
sTNFR [nglml]
Fig 4. Cowelation of circulating adhesion molecules and soluble
tumor necrosis factor receptor (sTNFR) (60 kd) levels in MS.
Since expression of adhesion molecules is upregulated by cytokines
such as tumor necrosis factor (TNFi-alpha. we examined the relationship between serum levels of shed L-selectin, VCAM-1,
and ICAM-1, and the circulating binding protein for TNFalpha, sTNFR. Regression analysis gave correlation coefficients
of 0.47 (p < 0.0001) far sL-selectin (A),0.36 (p < 0.0001)
for sVCAM-1 (B), and 0.28 (p < 0.0007) for slCAM-1
(C). ICAM-1 duta are from Hartung and colleagues {13).
lymphocyte clones was reported to be a prerequisite
for lymphocyte traffic to the brain and disease induction in the transfer EAE model 1271. Similar findings
were obtained with a panel of proteolipid proteinreactive T-cell lines 128). Binding of myelin basic protein-specific T-cell clone to brain endothelial cells
from newborn SJL mice in vitro was partially inhibited
by monoclonal antibodies to either VLA-4 or
VCAM-1 1291. Significantly, injection of monoclonal
antibodies to either alpha-4 beta- 1 integrin or
VCAM- 1 abrogated pathogenicity of the encephalitogenic T-cell clones in vivo 130).
Expression of L-selectin and VCAM-1 is induced or
greatly enhanced through the action of a number of
stimuli including lipopolysaccharide, interferon (IFN)
gamma, interleukin-1 (IL-l), and TNF-alpha (reviewed
previously [ 3 , 61). Among these, TNF-alpha may be
of particular importance since several lines of evidence
strongly implicate this cytohne in the pathogenesis of
MS [4, 51. TNF-alpha exhibits toxic properties toward
Hartung et al: Adhesion Molecules and sTNFR in MS
191
oligodendrocytes and myelin in vitro [31), it is present
in active inflammatory lesions of both EAE and MS [5],
and blockade of endogenously produced TNF-alpha
abrogates or attenuates EAE [32, 331. Increased levels
of TNF-alpha have been documented in the CSF of
MS patients and were strongly correlated to neurological impairment and CSF evidence of blood-brain barrier damage [12, 341. Finally, a rise in TNF-alpha production by blood mononuclear cells apparently
precedes clinical exacerbations [3 5-37]. TNF-alpha
mediates its biological effects through binding to two
distinct specific cell surface receptor types of 55- and
75-kd molecular size. Soluble forms that are derived
by proteolytic cleavage exist. Recently, increased levels
of the 60-kd sTNFR were detected in the serum of
patients with MS 1381. Hence, we chose to determine
serum concentrations of sTNFR levels and confirmed
this observation. sTNFR concentrations correlated
with levels of the circulating adhesion molecules sLselectin and sVCAM-1. While sTNFR may serve to
bind noxious TNF-alpha, it should be noted that there
are also suggestions that the biological half-life of TNFalpha may be prolonged after binding to the TNFR
c391.
Increased levels of the circulating adhesion molecules VCAM-1 and L-selectin probably reflect endothelial cell and lymphocyte/monocyte activation occurring in the setting of acute inflammatory processes
affecting the CNS. While these circulating forms initially were considered end products destined to clearance, there is now evidence that they may retain some
functional activity {4, 6, lo]. VCAM-1 and L-selectin
conceivably have a role beyond that in T-cell homing
and migration. They both serve as costimulatory signals
for T-cell activation and may therefore be important
accessory molecules for local initiation of autoaggressive T-cell responses in the CNS 125, 40). It is not
surprising that in other diseases also thought to be
caused by aberrant T-cell immune responses such as
rheumatoid arthritis, similarly elevated levels of these
adhesion molecules can be measured [7].
In acute relapses of MS or active progressive disease
and in certain viral encephalitides, production of cytokines such as TNF-alpha results in upregulation of adhesion molecules on endothelial cells and leukocytes.
Consequently, autoreactive T lymphocytes and activated leukocytes home to and invade the CNS to initiate local tissue damage. Shed adhesion molecules detectable in serum may serve as a disease marker,
although longitudinal studies are warranted to corroborate this notion. Results of a first study demonstrating
a close temporal association between clinical relapses
and circulating ICAM-1 levels during a 1-year period
with monthly sampling were published recently [41].
Apart from a yet to be proved role as surrogate marker
of disease activity, circulating adhesion molecules may
192 Annals of Neurology
Vol 38 N o 2
August 1995
also serve to downregulate ongoing inflammation. This
may also hold for sTNFR.
These observations along with those previously reported underscore the important role of adhesion molecules and TNF-alpha in the pathogenesis of MS and
highlight sites for future immunointervention. Blockade of immigration of autoreactive T cells in the CNS
could be achieved with antibodies to adhesion molecules, or possibly competitive ligands [30, 42). Similarly, TNF-alpha may be efficiently neutralized by administration of sTNFRs [43, 441. Clearly, appropriate
timing and dosing need to be worked out in detail
before such experimental therapy can be advocated.
Both approaches could advance more effective antigennonspecific treatment of MS.
This work was supported by the Bundesministerium fur Forschung
und Technologie (BMET 01KD9001).
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