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Elevated soluble interleukin-2 receptor levels in patients with active multiple sclerosis.

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Elevated Soluble Interleukm-2
Receptor Levels in Patients with Active
Multiple Sclerosis
Keiko Adachi, MD, Toshihide Kumamoto, MD, and Shukuro Araki, MD
~
~~
The level of soluble interleukin-2 receptor (sIL-2R) was quantitated with enzyme-linked immunosorbent assay in
serum and cerebrospinal fluid obtained from 24 patients with multiple sclerosis and 10 patients with other neurological disorders in whom immunological mechanisms are unlikely to participate. The sIG2R level in the serum and
cerebrospinal fluid of patients with multiple sclerosis in relapse was significantly higher compared with patients with
multiple sclerosis in remission and with controls. The sIL-2R level, especially in the cerebrospinal fluid, showed higher
sensitivity and specificity than other clinical parameters including the cerebrospinal fluid I g G ratio, peripheral lymphocyte CDWCD8 ratio, cerebrospinal fluid myelin basic protein and oligoclonal bands. Our data suggest that measurement of the sIG2R level may be useful in evaluating disease activity in patients with multiple sclerosis.
Adachi K, Kumamoto T, Araki S. Elevated soluble interleukin-2 receptor levels in patients
with active multiple sclerosis. Ann Neurol 1990;28:687-691
Some reports have indicated that multiple sclerosis
(MS) might be a T-cell-mediated immune disorder
[l]. Recent pathological studies showed that T lyrnphocytes expressing interleukin-2 (IL-2) and interleukin-2 receptor (IG2R) had infiltrated into demyelinated plaques in brains obtained at autopsy of
patients with active MS 12-41. Moreover, some authors have reported that serum IL-2 levels are elevated
in the active phase of MS and parallel the clinical
course 15-71. These findings strongly suggest that immune reactions may be closely related to the development of MS [lf.
Soluble IL-2 receptor (sIL-2R) is released from activated T cells together with IL-2 18, 91 and is increased in autoimmune disorders such as systemic
lupus erythematosus [101 and rheumatoid arthritis
1111. This finding suggests that quantitation of sIL-2R
may be useful for monitoring disease activity in autoimmune disorders.
To learn whether the levels of sIL-2R reflect the
clinical course of MS, we quantitated the sIL-2R level
in the serum and cerebrospinal fluid (CSF) of patients
with MS in relapse and remission, and compared its
sensitivity and specificity with other clinical parameters
112-141 including the CSF IgG ratio, peripheral lym-
From the First Department of Internal Medicine, Kumamoto University Medical School, Kumamoto, Japan.
Received Feb 6, 1990, and in revised form Apr 27. Accepted for
publication May 25, 1990.
phocyte CD4KD8 ratio, CSF myelin basic protein
(MBP) and oligoclonal bands.
Materials and Methods
Patients
Serum and CSF samples were collected from 24 patients
with clinically definite MS { 151, 15 women and 9 men with a
mean age of 35.1 years. All patients had the relapsingremitting form of the disease. Thirteen episodes of relapse
and 17 episodes of remission were analysed. We defined the
relapse phase as within 4 weeks after onset of clinical exacerbation, and the remission phase as from 5 weeks after onset
of clinical exacerbation. Control CSF samples were collected
from 10 patients with other neurological disorders in whom
immunological mechanisms are unlikely to participate, including ( 5 each) patients with muscle contraction headache
and mild psychoneurotic symptoms. Control serum samples
were collected from 10 healthy donors. CSF samples were
frozen immediately and stored at - 70°C until examined.
Routine CSF Studies
Albumin and I& concentrations in unconcentrated CSF and
corresponding serum were determined by the single radial
immunodiffusion technique 1163. IgG ratio in CSF (IgG/
albumin) was calculated. IgG oligoclonal band patterns in
concentrated CSF were demonstrated by agarose isoelectric
focusing followed by amido black (IOB) staining [I 7). MBP
Address correspondence to Dr Adachi, The First Department of
Internal Medicine, Kumamoto University Medical School, 1-1-1,
Hon103 Kumamoto 860' Japan'
Copyright 0 1990 by the American Neurological Association 687
0
MS
relapse
N=13
MS
controls
remission
N=15
MS
relapse
N=5
A
B
N=ll
MS
remission
N =10
controls
N=7
Fig 1. (Aj Serum levels of soluble interleukin-2 receptor (slL-2R)
in patients with multiple sclerosis (MS).Hatched columns and
horizontal bars indicate mean % SD. Closed circles indicate
samples from patients with steroid administration, and open circles indicate samples from patients without steroid administration. (B)The slL-2R levels in cerebrospinaljuid obtainedfmm
patients with MS. Hatched columns and horizontal bars indicate mean 2 SD. Closed circles indicate sampies from patients
with stwoid administration, and open circles indicate samples
from patients witholtt steroid administration.
in the CSF was quantitated by radioimrnunoassay r18). MBP
concentration and oligoclonal IgG band were examined only
in patients with MS.
Enzyme-linked Immunosorbent Assay for Soluble IL-2
Receptor and IL-2
CSF samples were concentrated to 20-fold at 4°C by using an
ultrafiltration apparatus, Minikon-B (B-15, Amicon, Danvers, MA). The sIL-2R levels were determined by the
enzyme-linked irnrnunosorbent assay (ELISA) by using the
IL-2R Bead Assay Kit (Cellfree, T Cell Sciences, Inc, Cambridge, MA) according to manufacturer instructions.
For the quantitation of IL-2 in biological fluids, a commercially available ELISA test (Intertest 2, Genzyme Corp, Boston, MA) with a slight modification was used. Serum and
CSF diluted 1:3 and 1:1, respectively, with phosphatebuffered saline solution were measured according to manufacturer instructions.
Statistics
The levels of IL-2 and slL-2R in CSF and serum were measured in duplicate, and the results were expressed as the
mean ? SD. IgG ratio and CD4/CD8 ratio were also expressed as the mean ? SD. Statistical analysis was performed
with the unpaired t test.
Results
The sIL-2R level in the serum of patients with active
MS in relapse was significantly higher than in those
patients in remission, and in disease controls (p <
0.01). As shown in Figure IA, the sIL-2R level in the
serum of controls, patients with MS in relapse, and
patients with MS in remission was 193.0 k 29.7,
361.2
125.8, and 229.6 2 74.7 U / d , respectively.
No significant difference was observed between patients in remission and disease controls. The sIL-2R
level in CSF from patients in relapse was 190.0 +356.0 Uiml, but it was below the detectable level in
patients in remission and in disease controls.
To compare sIL-2R levels in CSF with those in
serum, we calculated the ratio of sIG2R levels to albumin concentrations. The sIL-2R levels in CSF and
serum in relapse were 342.8 k 484.4 and 27.5 2 27.2
U/mg albumin, respectively. In CSF, sIG2R levels
were elevated about 12-fold higher than in serum. This
standardization also demonstrated that sIG2R levels in
CSF from relapsing patients were significantly elevated
compared with those from patients in remission and
disease controls @ < 0.05) (Fig 113). Steroid administration did not decrease sIL-2R levels in either the
serum or CSF.
As shown in Figures 2A and 2B, IL-2 levels in the
serum and CSF of disease controls were 2.2 +- 2.2 and
1.2 t 1.2 U/ml, whereas in patients with MS, these
values were 19.9 5 17.9 and 11.0 2 9.4 U/ml in
patients in relapse, and 2.5 L 5.1 and 0.4 k 0.8 Urn1
in patients in remission. Serum and CSF IL-2 levels
688 Annals of Neurology Vol 28 No 5 November 1990
,- PC0.05
5or
I
A
0
MS
MS
relapse
remission
N=10
N=17
controls
N=lO
Fig 2. (A) Serum levels of znterleukin-2 (IL-2) in patients with
multiple sclerosis (MS). Hatched columns and horizontal bars
indicate mean 2 SD. Closed circles zndicate samples from patients with steroid administration, and open circles indicate samples from patients without steroid administration. (B) The IL-2
levels in cerebrospinalfluid obtainedfrom patientj with MS.
Hatched columns and horizontal bars indicate mean & SD.
Closed circles indicate samples from patients with steroid administratzon, and open circles indicate samplcs from patients without
steroid adminzstration.
300
Y
N=9
controls
N=10
were significantly higher in patients in relapse than in
patients in remission @ < 0.05).
The IgG ratio in CSF from MS patients is shown in
Figure 3. The IgG ratios in CSF in relapsing patients
with MS, in remitting patients, and in controls were
23.9 & 6.896, 13.4 2 5.896, and 13.1 ? 2.1% respectively. In relapsing patients with MS, the ratio was
significantly higher than in remitting patients and controls @ < 0.01). In contrast, the CD4/CD8 ratios in
the peripheral blood lymphocytes were not significantly different (Fig 4).
An MBP value greater than 2 n g / d in the CSF was
observed in 4 of 9 relapsing patients with MS (4496),
while oligoclonal bands were detected in 6 of 14
0
g- 20-
0
U
v)
u
10 -
0
I
0
I
MS
MS
relapse
N=8
remission
N=8
ters, we calculated the rate of abnormal values (those
exceeding mean
2 SD of the controls). As shown in
the Table, increased CSF levels of sIL-2R were found
in all patients with MS with active-relapsing disease.
CSF sIL-2R showed the highest sensitivity and specificity of all the clinical parameters. Although less sensitive and specific than CSF values, sIL-2R in the
serum also showed good correlation with MS disease
activity. The coefficient value between sIL-2R levels in
the serum and CSF was 0.766 and was statistically significant (p < 0.01).
+
v)
~~
MS
remission
(43%).
To compare the sensitivity of sIL-2R and IL-2 in
relapsing patients with MS with other clinical parame-
.-C
$-m
R
MS
relapse
N=6
controls
N=9
~~
Fig 3. IgG ratio zn cerebrospznaljuid obtaznedfrom patients
wrth multzple sclerosis (MS).Hatched columns and horizontal
bars indzcate mean 2 SD. Closed circles zndzcate samples from
patwnts wrth steroid adminzstratzon, and open czrcles zndxate
samples from patzents wztbout rterozd admznzstratzon.
Discussion
In the present study, we found significantly higher
levels of sIL-2R and IL-2 in both the serum and CSF of
patients with MS in relapse compared with patients
with MS in remission, and in disease controls with
Adachi et
al:
Soluble IL-2R Levels in MS 689
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1 :.:
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1.0-
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Comparison of the Sensitivity of Soluble IL-2 Receptor and
IL-2 Levels with Other Clinical Parameters
0
0 : .
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0
:
0
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0
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:
“ 2 .:
0
0
0
Parameters
Soluble IL-2R
Serum
IL-2
CSF
0
0
0
:
0
:
1
1:
0
0
0
0
0
Fig 4. CD4iCD8 ratio in peripheral blood lymphocytes obtained
from patients with multiple sclerosis (MSI. Hatched columns and
horizontal bars indicate mean & SD. Closed circles indicate
samples from patients with stmid adminirtration, and open circles indicate samples from patients without .steroid adminijtration.
neurological disorders in whom immunological mechanisms are unlikely to participate. Recently, several
studies have reported correlation between IL-2 levels
in the serum and CSF, and disease activity of MS.
Gallo and colleagues [5] examined IL-2 levels in the
serum and CSF in the relapsing-remitting form of MS
in the active stage and in chronic progressive MS by
using the same ELISA kit we used. They recognized
that the IL-2 levels were statistically higher during
acute relapse than in the chronic progressive type of
MS. In contrast, Trotter and colleagues 161 observed
that serum I L 2 levels were higher in patients with
chronic progressive MS than in normal controls. Recently, Greenberg and coworkers [19] and Gallo and
colleagues [20] found that serum sIL-2R levels were
significantly higher in patients with MS than in normal
controls. Moreover, Gallo and colleagues {20] found
that none of the patients with MS showed a detectable
level of sIG2R in the CSF, although they detected IL-2
in the CSF in 11 of 50 patients with MS. They, however, used unconcentrated CSF, while in our present
study, we attempted to concentrate the CSF 20-fold.
All samples from patients with MS in relapse showed
detectable levels of sIL-2R in the concentrated CSF,
whereas no samples from 7 patients with MS in remission showed any increase.
The sIL-2R levels in the CSF closely reflected the
clinical course of MS. The sIL2R in the serum also
showed good correlation with MS activity, although it
Serum
CSF
IgG ratio (CSF)
CD4iCD8 (PBL)
MBP (> 2 ng)
OB (positive)
Relapse
(% Positive)
(sPositive)
11/13 (85)
11/11 (100)
5/15 (33)
0110 (0)
6/10 (60)
416 (67)
618 (75)
5/12 (42)
419 (44)
6/14 (43)
Remission
2/17 (12)
019 (0)
118 (13)
318 (38)
016 (0)
6114 (43)
With values greater than mean + 2 S D of controls defined to be
abnormal, the percentage of positive samples is calculated and depicted. An MBP value over 2 ngiml in the CSF is defined to be
positive. Detection of oligoclonal bands in the CSF is defined to be
positive. [IL-2 = interleukin-2; soluble IL-2R = soluble inter-
was less sensitive and specific than it was in the CSF
(see Table). Turning to other tests, the IgG ratio in the
CSF has been considered a reliable measure of MS
activity 1121. Indeed, 85% of our CSF samples from
active-relapsing patients with MS had ratios higher
than the mean + 2 SD, whereas only 20% of patients
with MS in remission did. MBP in CSF rises early in
MS exacerbations and is frequently back to normal by
2 weeks {l3]. In fact, more than 50% of our relapsing
patients with MS had normal CSF MBP, but sIL-2R
levels remained elevated up to 4 weeks after onset of
clinical exacerbation. Thus, it seems that sIL-2R level
in the CSF may be a more sensitive and specific parameter than the IgG ratio and MBP.
Previous data suggest that MS might be a T-cellmediated immune disorder. Indeed, perivascular lymphocyte infiltration expressing IL-2R [2-41 and Class I
and I1 major histocompatibility antigen (MHC)-positive astrocytes, acting as antigen-presenting cells, have
been found in active MS lesions [21]. Moreover,
decrease in the suppressorhducer subpopulation
(CD4+,2H4+) of peripheral lymphocytes [22], dysfunction of suppressor T lymphocytes 1231, and elevated IL-2 levels in the serum and CSF of patients with
MS during exacerbation 15, 61 have also been reported. Since sIL-2R is a product of activated T cells
{24}, OUT data support the idea that the immunological
mechanisms are closely related to the pathophysiology
of MS.
As shown in the present study, the sIL-2R level in
CSF closely reflects disease activity by clinical criteria.
Although less sensitive than sIL-2R measurement in
CSF, the sIL-2R level in serum also correlates well
with clinical disease activity. The coefficient value be-
690 Annals of Neurology Vol 28 No 5 November 1990
tween sIG2R levels in the serum and CSF was 0.766,
and was statistically significant @ < 0.01). Therefore,
sIG2R in serum is a simply done indicator of disease
activity, and may be useful in monitoring the effect of
treatment.
Supported by grants from the 1988 Neuroimmunological Disease
Research Committee, The Ministry of Health and Welfare of Japan.
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Adachi et al: Soluble IL-2R Levels in MS
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