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Cytokine profile of myelin basic proteinЧreactive T cells in multiple sclerosis and healthy individuals.

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Cytokine Profile of Myelin Basic ProteinReactive T Cells in Multiple Sclerosis
and Healthy Individuals
Guy Hermans, MS,* Piet Stinissen, PhD,* Lysiane Hauben, MS,* Ella Van den Berg-Loonen, PhD,?
Jef Raus, MD, PhD,* and Jingwu Zhang, MD, PhD*
Myelin basic protein (MBP)-reactive T cells have been implicated in the autoimmune pathogenesis of multiple sclerosis
(MS). In this study, we examined the cytokine profile of 531 primary MBP-reactive T-cell lines and 72 independently
established clones from 32 patients with MS and 18 healthy controls (NS) by using highly sensitive enzyme-linked
immunosorbent assays. An increased number of primary T-cell lines producing interferon-? (IFNy) and/or interleukin-4
(IL-4) in response to MBP were found in patients with MS compared with controls. No distinct T h l or Th2 subtypes
could be demonstrated among the MBP-reactive clones. IL-4 was more frequently observed among MS-derived clones.
Clones derived from MS patients produced increased levels of IL-2, IL-4, tumor necrosis factor-a (TNFa), IFNy, and
IL-10, but not IL-6. It is interesting that MBP-reactive T cells from MS patients expressing the disease-associated HLADRB1*15 allele produced increased quantities of TNFa, a cytokine suggested to play an important role in inflammation
and demyelination. When challenged with either MBP or a bacterial superantigen, the clones expressed similar levels of
the proinflammatory cytokine IFNy. Our study suggests a functional difference in T-cell responses to MBP in patients
with MS compared with healthy individuals, and provides further insights into the role of MBP-reactive T cells and their
cytokine profile in the inflammatory processes of MS.
Hermans G, Stinissen P, Hauben L, Van den Berg-Loonen E, Raus J, Zhang J. Cytokine profile of myelin basic
protein-reactive T cells in multiple sclerosis and healthy individuals. Ann Neurol 1997;42:18-27
-
-.
Various cytokines play an important role in the pathogenesis of autoimmune diseases. This is clearly illustrated in experimental allergic encephalomyelitis
( E M ) , an autoimmune animal model sharing many
similarities with multiple sclerosis (MS). EAE can be
induced in rodents by injecting myelin antigens such as
myelin basic protein (MBP) and by adoptive transfer of
MBP-reactive encephalitogenic T cells to naive syngeneic recipients [ 11. The encephalitogenicity of MBPreactive T cells is not only associated with their recognition of the encephalitogenic epitopes on MBP but
also with their cytokine profile. MBP-reactive T cells
capable of producing Thl cytokines were found to mediate EAE by adoptive transfer [2, 31. Furthermore, the
clinical course of EAE can be altered therapeutically by
regulating various cytokines in balancing the Thl and
Th2 cell ratio, indicating the role of these cytokines in
the autoimmune processes 14-61. Cytokines have also
been implicated in the pathogenesis of MS, a chronic
inflammatory disease of the central nervous system
(CNS) characterized by infiltration of inflammatory
cells including T lymphocytes in the brain lesions 171.
Although cytokines such as interferon-y (IFNy) are
considered to orchestrate the chronic inflammatory responses in MS, other cytokines such as tumor necrosis
factor-a (TNFa) were shown to possess demyelinating
potential [8]. The pathogenic role of T N F a is further
suggested by the demonstration that TNF-neutralizing
agents can prevent and suppress EAE [4, 51. Furthermore, some therapeutic interventions specifically manipulating the cytokine network were proved to be effective in the treatment of MS. Treatment with IFNB
reduces the relapse rate and brain lesions in relapsingremitting patients [9], although the mechanism of tolerance induction by oral feeding of MBP is considered
to partially involve induction of suppressive cytokines
1101.
There is increasing evidence suggesting that altered
T-cell responses to MBP play an important role in the
pathogenesis of MS (reviewed in References 11 and
12). Although MBP-reactive T cells are also present in
healthy individuals as part of the normal T-cell reper-
From the *Multiple Sclerosis Research Unit, D r L. WillemsInstituur and Limburgs Universitair Centrum, Diepenbeek, Belgium; and t'Tissue Typing Laboratory, Academisch Ziekenhuis,
Maastricht, The Netherlands.
Address correspondence to D r Herman?, MS Research Unit, Dr L.
Willems-Instituut, Universitaire Campus, B-3590 Diepenbeek, Belgium.
Received Aug 8, 1996, and in revised form Dec 3. Accepted for
publication Dec 5, 1996.
18
Copyright 0 1997 by the American Neurological Association
toire, they appear to undergo in vivo activation and
clonal expansion and accumulate in the brain compartment of patients with MS as opposed to healthy individuals [l3-191. It has been postulated that the altered
functional properties of MBP-reactive T cells may be
associated with the potential role of MBP-reactive T
cells in the pathogenesis of MS [13]. As demonstrated
in EAE, activated MBP-reactive T cells may acquire a
different pattern of cytokine production that enables
them to cross the blood-brain barrier by up-regulating the expression of major histocompatibility locus
(MHC) and adhesion molecules on the epithelium [20,
211. Thus, it is of considerable interest to elucidate the
cytokine production profile of MBP-reactive T cells
and its potential association with MS.
Murine T-helper lymphocytes can be categorized
into a Thl subset that mediates cellular immunity and
produces IFNy, TNFa, and interleukin-2 (IL-2), and
into a Th2 subset that is mainly involved in humoral
immune responses and secretes IL-4, IL-6, and IL-10
[22]. A third subset of murine Tho cells was shown to
produce a large array of cytokines, including IFNy,
TNFa, IL-2, IL-4, IL-6, and IL-10 [23]. Although
some human T-cell clones of various antigen specificities resemble muriiie ThlITh2 T cells in production of
the two distinct groups of cytokines, the cytokine profiles of human T cells are generally more obscure [24].
In this study, we examined the cytokine profile and
production of MBP-reactive T cells in patients with
MS and in healthy individuals. Two approaches were
taken to determine the specific cytokine production in
response to MBP stimulation. First, peripheral blood
mononuclear cells (PBMCs) were plated out in a microtiter plate format and stimulated with MBP to generate primary T-cell lines. The resulting cultures were
then assayed for specific production of the two reference cytokines IFNy and IL-4 after restimulation with
MBP to distinguish the Thl-, Th2-, and Tho-like subsets. In the second approach, a panel of MBP-reactive
T-cell clones were analyzed for production of IL-2,
IL-4, 1L-6, IL-10, IFNy, and TNFa. O u r data revealed a higher percentage of primary T-cell cultures
producing IFNy and/or IL-4 in response to MBP in
patients with MS as opposed to normal subjects (NS).
No clearly defined T h l , Th2, or Tho subsets were observed among the T-cell clones. IL-4 production was
more frequently observed among MS-derived primary
T-cell lines and clones. Quantitatively, MS-derived
MBP-reactive T cells secrete higher amounts of various cytokines, most significantly T N F a , IL-2, and
IL- 10. T N F a production was elevated significantly in
DRB1*15+ MS patients. Our results suggest functional differences in cytokine production between MSand NS-derived MBP-reactive T cells, which may be
related to their in vivo activation in patients with MS.
Materials and Methods
Cell Culture Media and Reugents
Cells were grown in RPMI 1640 culture medium supplemented with L-glutamine, sodium pyruvate, nonessential
amino acids, and 10 m M HEPES buffer (Life Technologirs,
Ghent, Belgium) and 10% heat-inactivated autologous human serum (Life Technologies). MBP was prepared and purified from normal human brain white matter according to
the method described by Deibler and colleagues [25]. Purity
was assessed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis and western blot analysis using a
mouse anti-human MBP monoclonal antibody (mAb) [26].
Tetanus toxoid (TT) was obtained from RIVM (Bilthoven,
The Netherlands).
Putients and Control Subjects
Peripheral blood samples were obtained from a total of 32
MS patients, consisting of 18 female and 14 male individuals; 20 of 32 were relapsing-remitting patients, and the remaining 12 patients showed a progressive disease course. The
mean age of the patients was 41 years (range, 22-57 years)
and the mean disease duration was 9 years (range, 1-20
years). Patients received no immunosuppressive treatments
during at least 2 months before blood sampling. Nineteen
healthy individuals, 15 females and 4 males, were randomly
selected. Their mean age was 41 years (range, 23-62 years).
Informed consent was obtained from all subjects volunteering for this study.
Generation of Antigen-Reactive Prirnury T-cell Lines
and Clones und Preparation of Culture Supernatants
for Cytokine Analysis
PBMCs were isolated from heparinized blood using FicollPaque density centrifugation (Pharmacia, Uppsala, Sweden)
and washed extensively with culture medium. For the generation of primary MBP-reactive or TT-reactive T-cell lines,
PBMCs were plated out at 1 X lo5 and 2 X lo5 cells/well
for MBP or 2 X 10' cells/well for TT (60 wells for each cell
density) in U-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark). Medium was supplemented with 10%
heat-inactivated autologous serum and contained 40 kg/ml
MBP or 20 Lf/ml TT. Cultures were kept in a humidified
incubator at 37°C and 5% CO,. After 7 days, culture supernatants were discarded from each well and cells were resuspended in fresh culture medium and split into two equal
aliquots. One aliquot was incubated with irradiated autologous PBMCs pulsed with the appropriate antigen (lo5 cells/
well) and the other with irradiated PBMCs pulsed with hen
egg lysozyme (HEL; Sigma, St Louis, M O ) as a control antigen. Supernatants were collected after 48 hours and evaluated in cytokine enzyme-linked immunosorbent assays
(ELISAs).
After removal of the culture supernatants, all MBP- or
TT-stimulated cultures received fresh medium supplemented
with 5 U/ml recombinant IL-2 (rIL-2) (Boehringer, Mannheim, Germany) and were cultured under the same conditions as described above. After an additional 5 days, each
culture was split and restimulated with MBP-pulsed autologous PBMCs or nonpulsed PBMCs ( l o 5 cells/well), respectively [ 131. Subsequently, cultures were incubated for 72
Hermans et al: Cytokine Profile of MBP-Reactive T Cells
19
hours and 1 pCi of 3H-labeled thymidine (Amersham,
Buckinghamshire, UK) was added 16 hours before the cell
harvest. Cells were harvested using an automated cell harvester (Betaplate 1295-004, Pharmacia) and ['Hlthymidine
incorporation was measured in a liquid scintillation counter
(Betaplate 1205, Pharmacia). A T-cell line was considered
MBP reactive when the mean cpm in duplicate wells containing MBP-pulsed PBMCs exceeded 1,000 and was greater
than the control cpm (in wells containing nonpulsed
PBMCs) by a factor of at least 3.
MBP-reactive T-cell clones were established using a previously described [13] cloning procedure in which cells were
seeded at 0.3 cell/well under limiting dilution conditions in
the presence of phytohemagglutinin (PHA; Difco, Detroit,
MI). Resulting clones were tested for their specific proliferative responses to MBP in proliferation assays as mentioned
above and were propagated by alternate rounds of stimulation with MBP and PHA in the presence of autologous
antigen-presenting cells to obtain sufficient amounts for further characterization.
Kinetics o f the Cytokine Production by
Antigen-Reactive T-Cell Clones
For the initial kinetics experiments, four MBP-reactive T-cell
clones were seeded at a density of 2 X l o 4 cells/well (Ubottom microtiter plates) in 200 yl of medium containing
10% heat-inactivated fetal calf serum. Cells were cultured in
two identical settings with irradiated MBP-pulsed autologous
PBMCs (lo5 cells/well) or nonpulsed PBMCs as a control.
Culture supernatants were harvested from separate wells after
24, 48, 72, 96, and 120 hours of incubation and the supernatants were pooled for cytokine analysis. [3H]Thymidine
incorporation was measured in parallel 3 days after stiniulation.
For cytokine production analysis and ['HI thymidine uptake measurements of MBP-stimulated T-cell clones, cells
were restimulated under similar conditions as described in
the kinetics section. For analysis of staphylococcal enterotoxin B (SEB)-induced proliferation and cytokine production, T-cell clones were stimulated by using 1 pg/ml SEB in
the presence of irradiated autologous PBMCs or by using
irradiated PBMCs alone as a negative control. An added negative control consisted of SEB-stimulated irradiated PBMCs
without T-cell clones. Conditioned supernatants and
[3H]thymidine-labeled parallel cultures were harvested after
72 hours of incubation in both types of experiments.
room temperature, with 50 (*.I of the matched biotinylated
detecting antibody (0.25 &ml mAb 860F10H12 and 0.16
pg/ml mAb 67F12A8 for IL-4 and IFNy, respectively, Medgenix) in BSA/PBS/Tween 20. Plates were then washed and
incubated with streptavidin-conjugated horseradish peroxidase (1: 1,500) (Jackson Immunoresearch Laboratories, West
Grove, PA) in BSA/PBS/Tween 20 for 30 niin. To develop
the color reaction, 100 pI of 3,3',5,5'-tetramethylbenzidinel
H,02 in citrate buffer (pH 5.0) was used as a substrate and
the reaction was stopped with 2 M H,SO,. Optical densities
were measured at 460 nm by using an ELISA reader, and the
concentrations of the respective cytokines in the sample were
caiculated by using a double eight-point standard curve of
the recombinant human cytokines (SL 58.128.10 and SL
58.123.10 for IL-4 and IFNy, respectively, Medgenix). The
lower limit of the linear range was less than 10 pg/ml for
both assays. A primary T-cell line was considered specific for
MBP when the cytokine production in duplicate wells containing MBP-pulsed PBMCs minus that in wells containing
HEL-pulsed PBMCs exceeded both the low limit of linear
range and the mean plus two standard deviations of the
intra-assay reproducibility.
To analyze the production of IL-2, IL-4, IL-6, 1L-10,
IFNy, and TNFa by MBP-reactive T-cell clones, commercially available ELISA kits were used according to the instructions of the manufacturer (Biosource, Camarillo, CA).
Detection limits varied between 8 and 16 pg/ml, depending
on the cytokine measured.
HLA Typing
Genotypes at the HLA loci DRB1, 3, 5, and 7 and D R Q l
were determined by allele-specific polymerase chain reaction
(PCR) on genornic DNA. Amplification products were visualized o n agarose gel electrophoresis by using ethidium bromide staining.
Statistics
T h e nonparametric Mann-Whitney U test was used to compare the mean cytokine production levels in MS and NS or
HLA allele+ and allele- groups because values were not
normally distributed [28]. analysis was used to compare
distribution differences between the groups. Correlation coefficients were calculated as Pearson product-moment correlation coefficients. Differences were considered significant
when p < 0.05. Data are presented as mean -C 1 standard
error of the mean (SEM).
x2
Cytokine Quantijfcation by ELLSA
Results
The production of 1L-4 and IFNy by primary lines was measured by using a sandwich ELISA with notineutralizing antibodies as a capturing agent [27].Microtiter plates (96-well)
(Imniunosorb F8, Nunc) were coated overnight at 4°C with
1 &mi of a mouse monoclonal antibody to human IL-4
( n A b 860A4B3; Medgenix, Fleurus, Belgium) or human
IFNy (mAb 350B10G6; Medgenix). Nonspecific binding
sites were saturated with 2% bovine serum albumin (BSA
Sigma) in phosphate-buffered saline (PBS)and subsequently
washed with a washing solution consisting of 0.02% Tween
20 in a 0.9% NaCl solution. Each supernatant sample (50
PI) was added and incubated concurrently, for 2 hours at
Kinetics of Cytokine Production o f MBP-Reuctive
T-Cell Clones
To determine the kinetics of cytokine production of
20
Annals of Neurology
Vol 42
NO 1 July 1997
MBP-reactive T-cell clones, four randomly selected
clones (MS-1.1 , MS-2.1, MS-23.1, and NS-1.1) were
stimulated with MBP-pulsed autologous PBMCs. IL-2,
IL-4, IL-6, IL-10, IFNy, and TNFa levels were measured in pooled culture supernatants obtained at different time points. Production was detected for all cytokines measured. As shown in Figure 1, production of
IFNy, IL-2, TNFa, and IL-6 reached their peak levels
250
800
f!,:
IL-2
200-
-
150-
400300-
200100
C
.c
0
1
2
3
4
5
6
0
1
2
3
4
5
0
1
2
3
4
5
6
0
1
2
3
4
5
I
I
I
1
I
1
2
3
4
5
0
0
3
'CI
2
Q
c
0,
z
- 0 1
- 0 9
6
0
6
Days of culture after stimulation
Fig 1. The kinetics of cytokine release by MBP-reactive T-cell clones. The kinetics of cytokine release of interleukin-.? (IL-2), IL-4,
ILL-6,IL-10, interfiron-y (IFNy), and tumor necrosis factor-a (TNFa) were examined by quantitative enzyme-linked immunosorbent assay. Four myelin basic protein (MBP)-reactive T-cell clones were stimulated with irradiated MBP-pulsed autologous peripheral blood mononuclear cells (PBMCs) or unpulsed irradiated PBMCs as a control. Data points represent mean cytokine concentrations in pooled supernatants of the clones minus background cytokine secretion levels in control wells. Background production levels
were below the sensitivig of the assayx used.
at 72 hours after stimulation with MBP. Although
maximal amounts of IL-4 and IL-10 occurred at 48
hours after stimulation, a substantial amount of both
cytokines was still present at 72 hours. Therefore, we
harvested the culture supernatants at 72 hours for cytokine detection in all subsequent experiments.
Frequency of Primavy MBP-Reactive T-cell Lines
To generate primary T-cell lines for cytokine analysis,
PBMCs were seeded in microtiter plates at predetermined optimal cell densities (200,000 and 100,000
cellslwell) and stimulated with MBP for 1 week. The
reactivity of each primary T-cell line to MBP was determined based on specific cytokine production as a
readout system (see Materials and Methods); 4,320 individual primary T-cell lines generated from 19 patients with MS and 17 healthy subjects were tested for
production of IFNy and IL-4 in response to MBP, using hen egg lysozyme (HEL) as a control antigen. A
total of 531 primary T-cell lines (319 of 2,160 lines
from MS and 212 of 2,160 from NS) were reactive to
MBP, producing substantially higher amounts of IFNy
and/or IL-4 after stimulation with MBP compared
with HEL. As shown on the left side of Figure 2, the
overall percentage of these cytokine-producing MBPreactive T-cell lines is higher in patients with MS than
in healthy subjects (14% vs 10%). The increased number of primary MBP-reactive lines in MS patients was
mainly due to T-cell lines producing IL-4. Because of
the large variations among individual data points, these
differences did not reach a statistically significant level
( p > 0.05).
It should be noted that when tested in proliferation
assays after an additional restimulation, only a small
fraction (31 of 531) of the cytokine-producing lines
showed a significant proliferative response (stimulation
index > 3) to MBP, and the percentage of MBPreactive T-cell lines tested by proliferation did not differ between MS patients and healthy controls (Fig 2,
right panel). The IL-4-producing T-cell lines lost their
responsiveness to MBP seven times more frequently in
the subsequent proliferation assays, compared with
Hermans et al: Cytokine Profile of MBP-Reactive T Cells
21
MBP-induced cytokine production at week 1
Specific proliferation
at week 2
0
0
0
0
**
0
0
t
o
0
m
o
*
0
0
"'R"'
0
***
...R.0 .. -
Fig 2. Percentage of early T-cell lines specifically secreting interferon-y (IFNy) or interleukin-4 (IL-4) in response to myelin basic
protein (MBP)). Peripheral blood mononuclear cells (PBMCs) were plated out at 200,000 and 100,000 cells/well (GO wells f i r each
cell densiq per subject) in the presence of MBP. Ajer 1 week, all cultures were split and stimulated with irradiated MBP-pulsed
PBMCs or unpulsed PBMCs, respectively. IFNy and IL-4 levels in culture supernatants were assessed ajer 48 hours by using quantitative enzyme-linked immunosorbent assays. The ley? side shows the percentage of primary T-cell lines secreting IFNy or ILL-4in
response to MBP, relative to a total of 120 wells tested (see Materials and Methods). All resulting primary MBP-stimulated lines
were expanded subsequently with recombinant IL-2 and tested f i r MBP-spec@ proliferation by f3H]tbymidine uptake at week 2.
Percentage of positive lines with a stimulation index > 3 is indicated on the right side.
those secreting IFNy. This selective loss was seen in
both MS- and NS-derived primary T-cell lines and in
parallel cultures using primary TT-reactive T-cell lines
(data not shown).
Cytokine Projle and Production
o j - Psimavy
MBP-Reactive T-cell Lines
Next, all MBP primary reactive T-cell lines were
subgrouped as Thl-like (IFNy+/IL-4-), Th2-like
(IFNy-/IL-4+), and Tho-like (IFNy+/IL-4+) cells. As
illustrated in Figure 3, a significant difference in the
distribution of Thl/Th2/ThO-like cells can be observed
between MS patients and controls (x' = 13.3, p <
0.002). Although primary MBP-reactive lines of the
Thl-like pattern dominated in both groups, the percentage of Th2-like primary lines was increased in MS
patients (22.3% vs 11.8%).
22
Annals of Neurology
Vol 42
No 1 July 1997
Quantitatively, the cytokine-secreting lines derived
from MS patients produced an increased amount of
IL-4 (78 ? 12 pg/ml vs 48 ? 7 pg/ml, p > 0.05). In
general, there was no correlation between the number
of IFNy producing and the number of IL-4 producing
lines within a given patient or control subject. Furthermore, no differences in cytokine production were observed between lines derived from patients with relapsing-remitting disease (13 of 19) and patients with
progressive disease (6 of 19).
Cytokine PsoJLile and Production of MBP-Reactive
T-cell Clones
A panel of 72 MBP-reactive T-cell clones was established from 16 patients with MS (9 relapsing-remitting
and 7 progressive patients) and 3 healthy controls. It
should be mentioned that these MBP-reactive T-cell
.-
‘--
I
87.7
MS
I
only IFNy (78% in NS vs 41% in MS). The differences in T h l - , Th2-, and Tho-like distribution between MS-derived and NS-derived clones were statistically significant
= 8.1, p < 0.02).
As shown in Table 2, with the exception of IL-6, all
cytokines analyzed (IL-2, IL-4, IL-10, T N F a , and
IFNy) were secreted in higher quantities by MBPreactive T-cell clones of MS patients compared with
NS. There is a significant correlation between the proliferation of the clones as measured by their stimulation
index and their cytokine production. The increased
IFNy and T N F a levels were positively correlated ( r =
0.359 and r = 0.343, respectively) with the proliferation of the clones. In contrast, the production of IL-10
correlated inversely with the proliferation ( r =
- 0.3 12).
(x’
TO
T 2
h
h
Cytokine profile of primary lines
T1
h
Fig 3. Cytokine profile of rnyelin busic protein-reactive prim a y T-cell lines derived from multiple sclerosis (MS) patients and normal subjects (NS). Cytokine patterns resembling
Tbl, Tb2, and TbO are dejned by selective production of
interferon-y (IFNy) (IFNyt/IL-4-), interleukin-4 (IL-4)
(“FNy- /IL-4+), or by mixed production of both cytokines
(IFNy+/IL-4+). Data are given as percentages of the primary
lines generated per group.
clones were derived from independent parental cell
lines, thus representing unrelated clonal origins. In the
first series of experiments shown in Figure 4, virtually
all 44 clones analyzed were found to produce IFNy
and T N F a . Most clones secreted IL-6 and IL-10,
whereas the pattern of IL-2 and IL-4 production was
rather heterogeneous. It is noteworthy that a significant
number of MS-derived clones produce IL-4, but only 1
of 9 clones generated from a healthy subject produced
a detectable amount of this cytokine. The overrepresentation of IL-4-producing clones among MS patients
was confirmed in our subsequent experiments in which
28 additional MBP-reactive T-cell clones (from 7 MS
patients and 2 healthy subjects) were tested selectively
for production of IL-2, IL-4, and IFNy (Table 1); 27
of 49 MS-derived clones secreted IL-4, whereas only 5
of 23 clones derived from healthy subjects produced
IL-4 ( p < 0.02). No differences were observed between clones derived from MS patients with relapsingremitting versus chronic progressive disease.
The cytokine profile of the clones was not comparable with the typical Thl and Th2 patterns observed in
murine models. However, using IFNy and IL-4 as reference cytokines, we classified all clones in T h l - , Th2-,
and Tho-like subtypes. As shown in Table 1 the distribution of the clones among the subtypes was different between MS patients and NS. Among the MSderived clones, an increased proportion expressed the
Tho-like phenotype producing both IL-4 and IFNy
(53% in MS vs 22% in NS). A high fraction of the
clones derived from 3 healthy individuals produced
Correlation of Cytokine Production and HLA
Haplotype o f the Patients
The alleles at the DRB and DQB loci were determined
for all patients and controls, and were correlated with
the cytokine production patterns of the respective
T-cell clones. Table 3 illustrates the increased TNFa
production by the MBP-reactive clones derived from
patients carrying the DRBl*15 or DRBl”07 alleles
compared with patients lacking these alleles. In contrast, T N F a levels were significantly lower in MS patients expressing the DRB l * l l or DRB l *03 alleles.
No significant HLA correlations were observed for
other cytokines.
Cytokine Profile of MBP-Reactive T-cell Clones aJter
Exposure to a Bacterial Superantigen
The activation route of MBP-reactive T cells in MS
remains unclear. Generally, it is believed that in the
initial stages of the disease the peripheral autoreactive
T cells are not activated by the CNS-derived antigens
such as MBP, but more likely by viral or bacterial
cross-reactive antigens or superantigens [ 11, 121. T o
further explore whether the cytokine profile of MBPreactive T-cell clones may vary when challenged with
different T-cell stimuli, including superantigens, we
stimulated 34 clones derived from 6 MS patients with
MBP or the superantigen staphylococcal enterotoxin B
(SEB). Proliferative responses induced by the stimuli
were correlated with the production of IFNy (data not
shown). Sixteen clones that responded to SEB stimulation expressed comparable levels of IFNy after exposure to MBP and SEB; 13 of these clones were studied
for T-cell receptor Vp gene expression and found to
express Vp17, V p l 2 , or Vp7 (data not shown). These
V p gene elements were previously reported to be associated with SEB reactivity [29, 301. Thus, production
of IFNy, a cytolune implicated in the disease process,
was comparable when the clones were stimulated with
MBP and with the superantigen SEB.
Hermans et al: Cytokine Profile of MBP-Reactive T Cells
23
v)
E
L
MS-10.1
9.2
MS-9.1
8.2
MS-8.1
7.4
7.3
7.2
MS-7.1
6.2
MS-6.1
5.4
5.3
5.2
MS-5.1
4.4
4.3
4.2
MS-4.1
3.8
3.7
3.6
3.5
L
I
c
II
I
:
I*
m
*
*
I
I*
0
600
1200
IBOC
l O
e
I
60
120
I:
0
1500
3000
0
600
300
IL-2
IFNy
.
.
.
0
600
1200
I
h
300
600
L
900, OF
I I:
300
600
300
600
0
Ik
.
1800
0
TNFa
60
120
0
IL-4
300
600
800
0
1
10
IL-10
IL-6
100
1000
SI
(pglml net production)
Fig 4. The cytokine profile of myelin basic protein (MB1')-reactive T-cell clones derived from multiple sclerosis (MS) patients and
normal subjects (NS). Cytokine concentrations in supernatants of MBP-reactive T-cell clones stimulated with MBP-pulsed autologous peripheral blood mononuclear cells (PBMCs) were measured after 72 hours of incubation. Background production o f cytokines
in the presence of unpulsed PBMCs was subtracted in rare cases where minor amounts of various cytokines were produced nonspeci$cally. Black bars represent net cytokine production. Stippled bars indicate proliferation as measured by stimulation index (SI) in
parallel experiments. Asterisks indicate cytokine concentrations below the linear range of the assay. N D = not determined; lFNy
= interfiron-y; IL
interleukin; TNFa = tumor necrosis fdctor-a.
1
Table I . Cytokine Profile of MS- and NS-Derived MBP-Reactive T-cell Clones
Group
n
Stimulus
IFNytIIL-4-
IFNyt/lL-4+
IFNy-IIL-4+
IFNy-IIL-4
MS
NS
Ratio of YoMSIYoNS clones"
49
23
MBP
MBP
20
18
0.5
26
5
1
0
2
0
2.4
-
_.
~.~
< 0.02.
a 2 -
x
-
8.1, D,
MS
=
multiple sclerosis; NS
=
normal subjects; MBP = myelin basic protein; IFNy = interferon-y; IL-4 = interleukin-4
Discussion
This study was undertaken to examine the cytokine
profile of MBP-reactive T cells in patients with MS
and in healthy subjects. As demonstrated in rodent
EAE models, the encephalitogenic properties of MBPreactive T cells are not only dependent on the epitope
recognition but are also related to their cytokine production. Production of T h l cytokines has been found
to be essential for the induction of EAE, and the bal-
24
Annals of Neurology
Vol 42
No 1 July 1997
ance between Thl and Th2 cells appears to alter the
clinical course of the disease [4-61. A similar mechanism may also be important in determining the pathologic potential of MBP-reactive T cells in MS. In this
study we analyzed the cytokine profile of MBP-reactive
T cells by using two approaches. First, primary T-cell
lines generated after a single stimulation with MBP
were tested for their cytokine production before adding
any exogenous cytokine to these cultures. This system
Table 2. Mean Cytokine Productioiz of MBP-Reactive T-cell Clones in Response to MBP
Groitp
IFNy
MS
Clones producingltested
Net production"
Clones producingltested
Net production"
Ratio of net cytokine
production between
two groups
NS
IL-2
46/49
25/40
1,434 2 187 218 i 63
23/23
6/15
142 i 77
812 i 83
1.5
1.8
TNFa
1L-4
IL-6
IL-10
34/35
833 -t 187
919
382 i 68
2.2
27149
95 f 30
5/23
80 i 26
1.2
27/35
8/34
398 2 98
252 -t 43
519
619
8 6 4 2 161 81 f 28
0.5
3.1
SI
49
50
13
23
68 2 18
0.7
*
"pg/ml, only considering clones producing the relevant cytokine.
MBP
= myelin basic protein; IFNy = interferon-y; IL
multiple sclerosis; NS = normal subjects.
=
interleukin; TNFa = tumor necrosis factor-a; SI
=
sriinulation index; MS =
Table 3. Correlation of TNFa Production and HLA Allele Expression in MS-Derived MBP-Reactive T-cell Clones
Allele-
Allele'
Allele
n
DRB 1*03
DRB 1*07
DRB1*11
DRB 1*15
21
5
12
14
TNFa
Production"
n
TNFa
Production"
Ratio
Allele+/Allele~~
P
561 i 170
2,012 i 909
329 t 86
1,041 It 367
15
30
23
14
1,117 i- 348
608 t 131
1,059 t 262
530 t 228
0.5
3.3
0.3
2.0
<0.04
<0.05
<0.04
c0.05
"pg/nil net production, mean 2 SEM.
TNFa
=
tumor necrosis factor-a; MS
=
multiple sclerosis.
may mimic the in vivo activation of resting MBPreactive T cells in MS patients. Second, the cytokine
pattern of a panel of established long-term T-cell
clones was studied.
Our study indicates a higher percentage of primary
T-cell lines producing IFNy and IL-4 after exposure to
MBP in patients with MS compared with healthy subjects. This contrasts with the previously reported frequency data that were based on [3H]thymidine uptake
data [13, 141. The discrepancy may be explained by
the increased number of IL-4-producing lines in MS,
combined with their selective loss before the ["]thymidine uptake assay is performed as shown here. Olsson and colleagues [31] also reported a higher frequency of MBP-reactive T cells in MS compared with
healthy controls, using a direct cytokine ELISPOT assay. The precursor frequency of MBP-reactive T cells
in the circulation as reported previously may therefore
be an underestimate of the actual in vivo frequency.
Thus, the current methods to generate MBP-reactive T
cells after repetitive in vitro stimulations may be biased
toward IL-4 populations, suggesting that the data obtained with these methods should be carefully evaluated.
The cytokine pattern of our MBP-reactive T-cell
clones does not resemble a typical Thl, Th2, or Tho
profle. No differences between MS patients and
healthy controls were obvious in the overall cytokine
pattern, with the notable exception of an increased
number of IL-4-producing clones in MS. It is interesting that proinflammatory cytokines such as IFNy and
TNFa are produced by virtually all clones examined.
These cytokines are known to induce M H C expression
on cells of the CNS, which may be important in perpetuating an ongoing inflammation 1201. TNFa has
been shown to be capable of directly killing oligodendrocytes in vitro [8]. In addition, local production of
these cytokines in the brain may recruit other cells to
the site of inflammation [4].It should be noted that all
the clones were isolated from the peripheral blood. It
remains to be studied whether MBP-reactive T cells
isolated from the cerebrospinal fluid (CSF), which may
be more relevant to the disease process, express a different cytokine production profile.
We observed increased T N F a production by the
clones that were isolated from DRBI*15+ MS patients. It remains to be studied whether this correlation
could explain the increased susceptibility of individuals
with this HLA allele in developing MS. The quantitative overproduction of TNFa in DRB1*15+ MS patients was also observed by measuring mRNA level by
using a quantitative PCR method (Vandevyver C,
Motmans K, Stinissen P, and co-workers, unpublished
data). Increased numbers of MBP-induced T N F and
Iymphotoxin mRNA-expressing mononuclear cells
were also demonstrated in the blood and CSF of MS
Hermans et al: Cytokine Profile of MBP-Reactive T Cells 25
patients [32]. Mutations or sequence variations within
or near the TNFa gene may be related to the altered
expression of the cytokine [33]. Although TNFa and
p gene polymorphisms were reported to be associated
with MS susceptibility in Swedish patients [34], no
such genetic association was found in another Swedish
study and a Belgian study [35, 361. The correlation
between TNFm overproduction and the HLA
DRB1*15 allele may prove to be important in the
pathogenesis of MS, but the current concepts do not
allow explanation of why other HLA alleles that are
not associated with the disease also appear to influence
the T N F a levels produced by the clones generated
from these patients. Further studies with clones of different antigen reactivity, and including genotypematched healthy controls, are necessary to reveal
whether the observation is also seen in healthy subjects
and whether this finding is antigen specific. However,
Zipp and associates [37] have reported TNFa overproduction by clones derived from control subjects with
the DRB1*15 allele.
Another observation with potential relevance to the
disease pathogenesis is our observation that stimulation
of MBP-reactive T-cell clones with a superantigen results in production of the proinflammatory cytokine
IFNy [29]. Furthermore, the pattern of cytokine secretion after superantigen stimulation closely resembles
that of antigenic stimulation. This finding adds further
evidence to the hypothesis that MBP-reactive T cells
may be activated nonspecifically in the peripheral circulation by superantigens in the early pathogenesis
[30]. Upon activation these cells may migrate to the
brain compartment by inducing MHC and adhesion
molecule expression on the blood-brain barrier endothelium through cytokine-mediated interactions. The
important role of IFNy in this interaction has already
been documented [20, 211.
In summary, abundant production of proinflammatory cytokines by MBP-reactive T cells both in MS and
in healthy controls was observed. An increased frequency of MBP-reactive T cells in the peripheral circulation of MS patients was noticed when using cytokine production as a readout system. Production of
TNFa is more abundant in MS patients expressing the
disease-linked DRB 1* 15 allele. Finally, superantigen
stimulation of these clones results in a similar cytokine
production profile, providing further evidence for the
putative involvement of such an event in the early
pathogenesis of MS.
This study was supported by funds from the “National Fonds voor
Wetenschappelijh Onderzoek,” the “Stichting Wctenschappelijk
Onderzoelc in Multiple Sclerose,” the “Limburgs Universitair Centrum,” the Belgian “Nationale Loterij,” the “Charcot Foundation
(Belgische studiegroep voor MS),” and the “European Concerted
Action on T Cell Auroimmui-rity in MS.” G.H. is a recipient of a
26
Annals of Neurology
Vol 42
No 1 July 1997
fellowship grant from the “Vlaams Instituut ter Bevordering van het
Wetenschappelijktechnologisch Onderzoek in de Industrie.”
Wc thank C. Bocken, K. Engelen, E. Smeyers, and D. Mercken for
their excellent technical assistance, D r M-P Jacobs for critical review
of the manuscript, and Drs R. Medaet, J. Nies, and G. den Hartog
for providing patient samples.
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Hermans et al: Cytokine Profile of MBP-Reactive
T Cells 27
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