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Cell-mediated immunity in multiple sclerosis as determined by sensitivity of different lymphocyte populations to various brain tissue antigens.

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ORIGINAL ARTICLES
Cell-Medated Immunity
in Multiple Sclerosis As Determined
by Sensitivity of Dfierent Lymphocyte
Populations to Various Brain Tissue Antigens
C. J. J. Brinkman, MD,* W. M. Nillesen," 0. R. Hommes, MD," K. J. B. Lamers, MSc,"
B. E. J. de Pauw, M D , t and P. Delmotte, PhDX
Peripheral blood lymphocytes from patients with multiple sclerosis (MS) or other neurological diseases and from
healthy individuals were separated by density gradient sedimentation into several subfractions. Individual cell
populations were cultured in the presence of several human brain tissue antigens. In comparison to controls,
mononuclear cells with a density of 1.077 gm/cm3from MS patients displayed a significantly increased sensitivity
after incubation with purified human myelin basic protein (MBP) but not with other brain tissue antigens. In
particular, the lymphocytes of patients suffering from MS for more than four years reacted positively with MBP,
suggesting that the reaction was time dependent. No difference between MS patients and controls in sensitivity to
any brain tissue antigen could be detected with cells of lower density (i.e., 1.073 to 1.069 gm/cm3 or < 1.069
gm/cm3). Comparable lymphocyte activity was found to antigens isolated from both MS and control brain tissue.
These results suggest that patients with chronic progressive MS have a secondary immune activity to MBP.
Brinkman CJJ, Nillesen WM, Hommes OR, Lamers KJB, de Pauw BEJ, Delmotte P: Cell-mediated
immunity in multiple sclerosis as determined by sensitivity of different lymphocyte populations
to various brain tissue antigens. Ann Neurol 11:450-455, 1982
Contradictory results have been reported concerning
cellular immunity during the course of multiple
sclerosis (MS). Lymphocyte stimulation tests have
been used in many studies on the immunopathology
of MS. Lymphocytes sensitized to purified myelin
basic protein (MBP) have been demonstrated in the
peripheral blood of MS patients by some investigators but not by others (discussed in [4, 5, 9, 12,
241). Furthermore, responses to MBP have been
found using lymphocytes from patients with other
neurological diseases ( O N D ) and cells from healthy
individuals [ I 1, 121, and therefore this phenomenon
is considered nonspecific. As suggested earlier by
Knight [ 131, variable technical conditions influencing
the sensitivity of the assay system may partly explain
these discrepancies.
Low-density lymphocytes are more reactive to
stimulants than are high-density lymphocytes [22].
Consequently, selection of human lymphocytes by
density before incubation with the antigens might
enhance the sensitivity of the test system. The goal of
the present investigation was to compare the responses to several brain tissue antigens of lymphocytes with varying density from MS patients to those
from controls.
From the Departments of "Neurology and +Hematology, Radboud University Hospital, Reinier Postlaan 4, 6525 GC Nijmegen, The Netherlands, and the $National Centnun voor Multipele Sclerose, Vanheylenstraat 16, 1910 Melsbroek, Belgium.
Received Mar 13, 1981,and in revised form June 25. Accepted for
publication July 13, 1981.
Materials and Methods
Sixteen patients (mean age, 37 years; range, 20 to 60 years)
suffering from the chronic progressive form of MS
(diagnosed according to the criteria described by
Schumacher et a1 [21]) were studied. Patients were hospitalized to undergo clinical and laboratory investigations.
During this period they did not receive immunosuppressive therapy. Degree of disabtlity was measured by a
disability standard score evaluation. The standard neurological examination was used to quantify neurological
signs [lo]. Controls consisted of 16 healthy individuals
(mean age, 31 years; range, 22 to 5 5 years) and 20 patients
suffering from OND (mean age, 41 years; range, 20 to 80
years), as follows: cerebrovascular disease, 7 patients;
epilepsy, 2 patients; lumbar disc herniation, 3 patients; and
normal pressure hydrocephalus, vitamin deficiency, basilar
impression, Guillain-BarrP syndrome, astrocytoma, Alz-
Address reprint requests to D~ ~
450 0364-5 134/82/050450-06$0l.25 @ 1981 by the American Neurological Association
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heimer disease, acoustic neurinoma, and trauma, 1 patient
each.
Cerebral tissue of a patient who suffered from chronic
progressive MS and of another who died from liver cirrhosis (control) were isolated within 6 hours after death
and used as antigen sources.
All further procedures were carried out at 0 to 4°C.
White matter was separated from gray matter. Both tissue
fractions were homogenized individually in a PotterElvehjem homogenizer in 0.01 M ammonium acetate, p H
7.2. The suspensions were sonicated until homogeneous
mixtures were obtained. The mixtures were centrifuged at
15,000 g for 30 minutes. Supernatants were lyophilized
and the products stored at -20°C until used. The antigen
fractions were designated SW (soluble fraction of white
matter) and SG (soluble fraction of gray matter). The
amount of protein in each fraction, based on dry weight material, was about 50% [16]. The precipitates obtained after
centrifugation were washed with 0.01 M ammonium acetate, p H 7.2, by centrifugation at 15,000 g for 30 minutes
until the supernatants remained protein free. The precipitates were lyophilized and stored at -20°C until used. The
insoluble white and gray matter brain tissue fractions were
designated IW and IG, respectively. MBP was isolated
from normal human brain tissue as previously described
[61.
Defibrinated venous blood was mixed with an equal volume of Tris-buffered minimal essential medium (MEM),
p H 7.4. Lymphocytes were isolated by centrifugation with
Ficoll-Isopaque (density, 1.077 gm/ml) [7] and washed
twice with MEM. A sample of this mononuclear cell suspension was cultured with various antigen fractions, and
the remainder was layered upon a discontinuous FicollIsopaque gradient with depsities of 1.073 and 1.069 gm/ml.
The separation of cells with different densities has been
described previously [ 151. Individual lymphocyte fractions
were washed twice with MEM and cultured with the various antigen fractions, depending upon the yield of cells.
The culture medium consisted of 1 ml of MEM supplemented with 20% type A (rhesus positive) heat-inactivated
human serum, 100 units of penicillin, and 100 pg of streptomycin. The lymphocytes were cultured in triplicate in a
concentration of 3 x los cells per milliliter over 6 to 7 days
at 37°C. White and gray matter fractions were added in the
amounts of 250 pg dry weight material for SW and SG and
100 pg dry weight material for IW and IG. According to
preliminary experiments, both doses would give optimal lymphocyte responses. Ten micrograms of MBP was
added. Sterility of cultures was controlled by incubation of
the culture medium lacking lymphocytes with the antigen
fractions. Cell viability 'was always checked .by incubating
the lymphocytes with 25 pg of pokeweed mitogen.
Twenty-four hours before the cells were harvested by
filtration under reduced pressure by means of a
semiautomatic harvester, 0.6 pCi of 3H-labeled thymidine
(specific activity, 25 to 30 Ci/mmol; Amersham, England)
was added. The dried filters were incubated in 6 ml of
Lipoluma, and radioactivity was counted in a liquid scintillation counter. The degree of lymphocyte activity was expressed as the amount of radioactivity incorporated into
lymphocytes cultured in the presence of antigen or mito-
gen minus the spontaneously incorporated "-labeled
thymidine (net cpm). In the case of MBP, we also calculated the stimulation index (SI) according to the following
formula:
SI
=
cpm measured in lymphocytes cultured with MBP
cpm measured in lymphocytes cultured without MBP
The Kruskal-Wallis test and the Mann-Whitney U-test
were used for statistical evaluation of results. Differences
0.05.
were considered significant at p
Results
The limited yield of mononuclear cells from some
individuals was responsible for the varying amounts
of data in the figure and tables. In o r d e r to compare
the antigenicity of MS brain tissue to control brain
1.077 gm/cm3) from
tissue, lymphocytes (density
14 MS patients and 18 OND patients were cultured
with the soluble or insoluble fractions of white and
gray matter isolated from both types of brain tissue,
as described previously. No significant differences ( p
> 0.05) could be detected between corresponding
antigenic fragments isolated from MS and control
brain tissue in their potency to stimulate lymphocytes
of either patient group. Therefore, only the results
obtained with the tissue parts from the control brain
are given.
Table 1 compares t h e responses of various lymphocyte subfractions, separated according t o density, of
MS patients, OND patients, and healthy individuals
t o t h e different antigen fractions. As demonstrated
in that table, purified MBP was the only antigenic
fragment that was of importance. A nearly significant
difference ( p = 0.07, Kruskal-Wallis test) was found
in lymphocyte activity (expressed as net cpm) between MS patients, OND patients, and healthy individuals following incubation of their nonfraction1.077 gm/
ated lymphocyte population (density
cm3) with MBP.
The difference in activity expressed as net cpm
might be influenced by individual differences in
spontaneous lymphoproliferation. Therefore, the
stimulation index was also used as an indicator of antigenic stimulation. In this way a significant difference ( p = 0.03, Kruskal-Wallis test) was found
among the three groups studied following incubation
of their nonfractionated cells with MBP, b u t n o t with
cells of lower density (Figure).
In Table 2 t h e MBP sensitivity is analyzed statistically using t h e Mann-Whitney U-test. Incubation of
t h e nonfractionated lymphocyte population (density
1.077 gm/cm3) with MBP resulted in significantly
increased incorporation of 3H-labeled thymidine
(expressed as net cpm or as SI) into the cells of MS
patients compared with OND patients or healthy
Brinkman et al: Cell-mediated Immunity in MS
451
Table 1 . Responses of Various Lymphocyte Density Subfractions t o Fractions Isolated
from Human White or Gray Matter and Myelin Basic Proteina
Cell Density
(gm/cm3)
S
Tissue
Fraction
Multiple
sw
1,897
3,168
2,702
2,528
1,499
2,840
2,305
2,878
290
1.077
(16)
(14)
(14)
(14)
(16)
(8)
(7)
(7)
(6)
19 (12)
191 (7)
2,291 ( 5 )
797 (5)
563 (3)
1,471 (14)
SG
IW
IG
MBP
sw
1.073-1.069
SG
IW
IG
MBP
G
sw
1.069
Healthy
Individuals
Sclerosis
SG
IW
IG
MBP
2,565
3,893
776
1,399
58
2,178
2,751
-404
1,855
-2
9,032
4,430
2,893
4,664
534
O t h e r Neurological Diseases
(16)
(16)
(16)
(16)
(16)
(11)
(11)
(11)
(10)
(13)
(9)
(8)
(7)
(5)
(10)
2,949
1,933
232
181
6
2,315
2,413
210
1,611
-136
13,661
10,730
0
-56
68
Significanceb
p
(20)
(20)
(20)
(20)
(20)
(11)
(10)
(8)
(8)
(14)
(11)
=
(9)
(6)
(5)
(11)
0.97
0.67
0.28
0.23
0.07
0.95
0.99
0.47
0.75
0.83
0.37
0.79
0.94
0.59
0.72
"Data represent median net cpm. Numbers of patients are given in parentheses.
bKruskal-Wallis test (statistical comparison of three groups together).
SW = soluble white, SG
basic protein.
= soluble gray,
1W = insoluble white, IG
=
insoluble gray matter, all ofcontrol brain tissue; MBP
-
1.073 1069 g I c ? ~
21k1.077g/crn3
=
human myelin
,1.069gIcm3
b
:1 3
12.
11..
10..
X
43
.-C
C
9..
8..
0
.-
5
z
7.
.- 6.
c
u)
5..
4..
"
3..
8.
2-,
'1
0
:-
v
0
HI
OND
MS
P, 0 03
:
..
k
k
HI
OND
P, 0.78
i
i
MS
:
r
i
HI
OND
..I
i
MS
P =o a 7
Stimulation of lymphocyte density subfractions from patients
with multiple sclerosis (MS),patients with other neurological
diseases ( O N D ) , and healthy individuals ( H I ) i n response to
myelin basic protein. The p values were obtained by testing the
three groups together (Kruska-Wallis test).
452
Annals of Neurology
Vol 11 No 5 May 1982
Table 2. Statistical Differences between Subject Groups i n Sensitivity of Varioas
Lymphocyte Density Subfractions to Myelin Basic Protein a
Cell Density
inm/cm3)
4
1.077
1.073- 1.069
1.069
Significance between:
Determination
MS-HI
MS-OND
OND-HI
Net cpm
SI
0.05
0.04
0.62
0.68
0.76
0.68
0.04
0.70
0.47
0.57
0.86
0.48
0.64
N e t cpm
SI
Net cpm
SI
0.01
0.95
0.47
0.51
0.84
aData represent p values (Mann-Whitney U-test). Significance between individual groups was calculated using net cpm values as well as
stimulation indices.
SI = stimulation index; HI = healthy individuals.
Table 3. Differences i n Lymphocyte Sensitivity to Human Myelin Basic Protein between Patient3
with Multiple Sclerosis for Shorter or Longer than Indicated Duration"
Duration
(Yr)
3
4
5
6
8
Shorter
1.00
1.40
1.50
1.75
1.80
(N
4)
(N = 6)
(N = 7)
(N = 8 )
(N = 1 1 )
=
Longer
Significanceh
2.05
2.25
2.10
2.25
2.40
p
( N = 12)
( N = 10)
( N = 9)
(N = 8)
(N
=
=
0.13
0.06
0.12
0.13
0.08
5
1.077 gm/cm3.
5)
aValues represent median stimulation indices. Numbers of patients are given in parentheses. Lymphocyte density was
bMann-Whitney U-test (one-sided).
Table 4. Statistical Comparison between Subject Groups i n Sensitivity of Nonfractionated
Lymphocyte Population t o Different Brain Tissue Fractions a
Significance between:
Tissue Fraction
MS-L-HI
MS-L-OND
MS-R-HI
MS-R-OND
M B P (net cpm)
0.02
0.007
0.63
0.80
0.15
0.14
0.02
0.004
0.53
0.61
0.04
0.08
0.50
0.79
0.4 1
0.55
0.76
0.77
0.42
0.4 1
0.67
0.75
0.7 1
0.37
MBP (SI)
sw
SG
IW
IG
aData represent p values (Mann-Whitney U-test). Lymphocyte density was 1.077 gm/cm3.
MS-L, MS-R = patients suffering from multiple sclerosis for more or less than four years, respectively; HI = healthy individuals; O N D =
patients with other neurological diseases; MBP = human myelin basic protein; SW = soluble white; SG = soluble gray, IW = insoluble
white, IG = insoluble gray matter, all of control brain tissue.
persons. With cells of lower density we could not
detect any difference in MBP sensitivity between MS
patients and controls.
The duration of the disease appeared to influence
the degree of sensitivity to MBP (Table 3). The
largest difference, nearly reaching statistical significance, in stimulation of lymphocytes (density
S 1.077 gm/cm3) by MBP was between patients
suffering from MS for more than four years (MS-L)
compared with those having the disease for less than
four years (MS-R). With MBP stimulation, lympho-
cytes from MS-L patients showed increased activity
compared with cells from OND patients or healthy
persons (Table 4). This phenomenon could be detected only with the nonfractionated lymphocyte
population (density
1.077 gm/cm3). A significant
difference was also observed between MS-L patients
and OND patients regarding their lymphocyte sensitivity to the IW fraction. None of the antigenic substances caused significant differences in uptake of
3H-labeled thymidine between cells of MS-R patients
and OND patients or healthy persons with any lym-
Brinkman et al: Cell-mediated Immunity in MS
453
phocyte subfraction tested (see Table 4). No relation
was found between the presence of a cell-mediated
immune response to MBP and the disability of MS
patients (as judged from their disability standard
score) o r the severity of their disease (as assessed on
neurological examination).
Discussion
The presence of oligoclonal immunoglobulin fractions and elevated immunoglobulin concentration in
the cerebrospinal fluid of MS patients are among several findings that point to altered immunocompetence in patients with MS [ 2 5 ] . Contradictory results
have been published concerning involvement of the
cellular immune system in the pathological process
underlying MS.
The results of the present study demonstrate increased sensitivity of the unfractionated lymphocyte
population to purified MBP in patients with chronic
progressive MS in comparison with controls. It is
tempting to assume, at least in our test system, a need
for cooperation between different types of lymphocytes in order to obtain adequate stimulation by
MBP. Alternatively, the presence in the peripheral
blood of MS patients of MBP-sensitized lymphocytes
with a density between 1.077 and 1.073 gm/cm3 may
be responsible for this phenomenon. Our failure to
detect a difference in immune response to MBP between MS patients and controls with low-density
lymphocytes ( s 1.069 gm/cm3)), however, may be
based on the large number of monocytes that are
predominantly encountered in the low-density fraction.
Because of the possibility that the MBP-induced
lymphocyte proliferation response in individual
subjects may be influenced by differences in spontaneous activity of lymphocytes [18], both net cpm
values and stimulation indices have been used as
indicators for reactivity to MBP. Despite slight differences in statistical analysis, both sets of data
pointed to similar conclusions. Moreover, no differences between the groups were observed in their
spontaneous or mitogen-driven lymphocyte proliferation with either cell fraction (Brinkman et al, in
press). The difference in lymphocyte sensitivity between persons with MS and O N D patients or healthy
individuals appeared to be restricted to MBP because
no differences were found among the three groups
when other central nervous tissue antigens were
used.
Polyacrylamide gel electrophoresis of the soluble
fraction of gray and white matter used in this study
showed no protein bands in the MBP position. The
failure to detect an increased cellular immune response in MS patients using insoluble white matter,
which consists mainly of membranous particles of
454 Annals of Neurology Vol 11 No 5 May 1982
oligodendrocytes, suggests that MBP is hidden in
these cell wall vesicles or else that structural differences resulting in a change of antigenicity exist
between released and membrane-bound MBP. This
might explain why we observed a difference in sensitivity to insoluble white matter between OND patients and patients who had suffered from MS for
more than four years, although the difference in
comparison to healthy persons was not significant.
Patients with longstanding MS appeared to be more
strongly sensitized than patients with MS of short
duration. An earlier study showed increased percentage lymphoblastic transformation in response to
human white matter in peripheral blood leukocyte
cultures of persons with MS compared to control patients [2]. In that study the investigators used a
supernatant of homogenized white matter, which was
obtained after centrifugation at rather low speed and
consequently might have contained antigenic fragments similar to those present in our insoluble white
matter fraction.
No significant difference in the ability to stimulate lymphocytes could be observed between white
or gray matter fragments isolated from brain tissue of
a patient with MS and corresponding fragments from
a patient presumably without damage to the central
nervous system. As compared to control brain
homogenates, however, MS brain homogenates
showed an enhancing effect on the active E-rosette
forming lymphocytes of MS patients [19, 20, 261.
This phenomenon is probably caused by non-MBP
constituents, because MBP isolated from MS brain
tissue appears to be chemically normal [3, 281. More
studies using different techniques are needed to
permit definite conclusions concerning an immunological relationship between MS brain extracts
and normal brain material.
The question of whether the immune response to
MBP is a primary event or only a consequence of
brain destruction by other unknown causes is still
hard to answer. Increased cell-mediated immune activity to MBP [23] and to human white matter [171,
as measured with the migration inhibition test, could
be demonstrated in MS patients preceding an
exacerbation. In accordance with earlier studies [ 11,
121, we were able to show that disease duration positively influenced the degree of sensitivity to MBP,
although other investigators found the greatest
lymphocyte reactivity to MBP in patients with MS of
short duration [91.
This presumable time dependency points to a secondary phenomenon which seems not to be
influenced by the degree of disability [9] or severity
of disease. A positive correlation between MBP sensitivity in MS patients and the severity or stage of
disease was described by others [8]. One might
speculate about the role of the MBP-sensitized lymphocytes predominantly in patients suffering from
MS for a longer time, as was found in this investigation. The present study comprised only patients who
suffered from a chronic progressive form of MS. Elevated numbers of human MBP-binding mononuclear
cells were detected predominantly in patients suffering from chronic progressive MS for more than
four years [27]. Lisak and Zweiman [14] found increased reactivity to MBP by peripheral blood lymphocytes as well as lymphocytes isolated from the
cerebrospinal fluid of patients with progressive MS,
but not by lymphocytes of patients with a stable form
of the disease. Patients with progressive MS showed
a relative deficit of suppressor T-lymphocytes in their
peripheral blood as compared to MS patients in remission or healthy controls [ 11. This relative deficit of
suppressor cells might be responsible for the detection of MBP-sensitized lymphocytes in vitro as described in this study. Moreover, the diminished
number of suppressor T-cells could allow MBPsensitized lymphocytes to influence the progression
of the disease. Experiments to investigate this hypothesis are in progress.
~~
Supported by the Princess Beatrix Foundation.
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455
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