Full text of DOI:10.1177/135245850000600501

Multiple Sclerosis
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pattern of cytokine secretion by peripheral blood cells of patients with multiple sclerosis in Brazil
Patrícia Mara da Costa, Clarissa Lin Yasuda, Silvia M Scagliusi, Blanca Maria Diaz-Bardales, Ernane Maciel, Benito P
Damasceno, M Heloisa SL Blotta, Charles P Tilbery and Leonilda MB Santos
Mult Scler 2000 6: 293
DOI: 10.1177/135245850000600501
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Multiple Sclerosis (2000) 6, 293±299
2000 Macmillan Publishers Ltd All rights reserved 1352 ± 4585/00 $15.00
ã
www.nature.com/ms
Pattern of cytokine secretion by peripheral blood cells of patients with
multiple sclerosis in Brazil
PatrõÂcia Mara da Costa1, Clarissa Lin Yasuda1, Silvia M Scagliusi1, Blanca Maria Diaz-Bardales1,
Ernane Maciel2, Benito P Damasceno2, M Heloisa SL Blotta3, Charles P Tilbery4 and
Leonilda MB Santos*,1
2
1Department of Microbiology and Immunology, Institute of Biology, UNICAMP, Campinas, Brazil; Department of
3
Neurology, Medical School, UNICAMP, Campinas, Brazil; Department Clinical Pathology, University of Campinas,
4
UNICAMP, Campinas, Brazil; Department of Neurology, Medical School, Santa Casa de SaÄo Paulo, SP-Brazil
Autoimmune T cells play a key role as regulators and effectors of organ-speci®c autoimmune disease. In multiple sclerosis (MS), activated T cells
speci®c for myelin components produce a plethora of in¯ammatory cytokines and mediators that contribute to myelin damage. The production of
proin¯ammatory and regulatory cytokines by peripheral blood cells from patients with active and stable MS and healthy controls were examined.
The results show that TNFa production was somewhat elevated in active MS with no signi®cant increase in the level IFNg, whereas in the chronic
phase the anti-in¯ammatory cytokines IL-10 and TGFb increased, accompanied by a reduction in IFNg when stimulated by myelin basic protein.
Multiple Sclerosis (2000) 6, 293±299
Keywords: cytokines; myelin basic protein; autoimmunity
Introduction
Multiple sclerosis (MS) is the most important demye-
linating disease that affects man. One feature which
distinguishes MS from most other conditions is its
geographic distribution. The geographic pattern based
on prevalence surveys suggests that rates are high in
southern Canada and the northern part of the United
States, but appear to be low in Mexico and Central
America. Few reliable data from Latin America are
available, however, and there is a need for information
about South America in particular. Although little
information is available about the prevalence of MS in
Brazil, specialists are concerned with its increase in
southern parts of the country, where the population
consists mainly of descendents of European immi-
grants.
Multiple sclerosis is a chronic in¯ammatory disease
characterized by lymphocyte in®ltration and demyeli-
nation of the central nervous system.1 Autoreactive T
cells which exist in an activated state in the peripheral
blood and accumulate in the cerebrospinal ¯uid (CSF)
of patients with MS2 are thought to recognize myelin
components such as myelin basic protein (MBP) and
thus contribute to the pathogenesis of the disease.
Cytokines play a particularly important role in cell
immune mechanisms. The investigation of the role of
cytokines in autoimmune diseases has expanded
rapidly over the past few years, and there is now
considerable evidence that cytokines such as TNFa
and IFNg contribute to the pathogenesis of MS. The
in¯ammatory responses caused by Th1 cytokines may
be inhibited by the emergence of a Th2 type response,
resulting in the down regulation of the Th1-mediated
immune response. Signi®cantly higher levels of TNFa
were found in the CSF of patients with chronic
progressive MS than in that of patients with a stable
form of the disease.3 The level of TNFa also correlated
with the degree of disability in patients with
progressive forms of the disease,4 and it has been
suggested that the increased production of TNFa
precedes the clinical manifestations of MS relapse.5
A
pathogenetic role for IFNg has also been
demonstrated in a clinical trial which revealed its
enhancement of exacerbation frequency in MS pa-
tients, possibly associated with the activation of
monocytes, as evidenced by up-regulation of MHC
class II antigen expression on these cells.6 IFNg also
stimulates the expression of adhesion molecules on
endothelial cells, promoting
T cell homing and
activating blood-derived macrophages and astrocytes
to produce proin¯ammatory mediators such as
TNFa.7,8
It has become apparent that in in¯ammatory
diseases involving the increased production of cyto-
kines with proin¯ammatory potential, there is
a
concomitant increase in production of cytokines with
immunoregulatory properties such as ILH 10 and
TGFb.9 ± 11 IL-10 was initially identi®ed as a factor
produced by
T helper 2 inhibiting the cytokine
synthesis of Th1, an effect attributed to the inhibition
of the accessory function, including downregulation of
MHC class II expression, and leading to impaired
antigen presentation to reactive T cells.12 ± 14
Several studies indicate that TGFb is a potent
immunosuppressive factor, affecting the proliferation,
*Correspondence: LMB Santos
Received 27 April 1999, accepted 12 June 2000
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Changes in patterns of cytokines in relapsing and remitting MS
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294
activation and differentiation of the cells that partici-
pate in immunity. Treatment of rats or mice with TGFb
ameliorates Experimental Autoimmune Encephalo-
myelitis (EAE),10 and the appearance of TGFb in the
CNS is related to recovery.15
In the present study, pro-in¯ammatory and anti-
in¯ammatory cytokines from the peripheral blood cells
of patients with active MS and from those in remission
were quanti®ed.
incubation at 48C, the plates were washed three times
with ELISA wash buffer (PBS containing 0.05% Tween
20, 0.001% Thimerosal) and blocked for 1 h with
ELISA diluent (PBS containing 0.05% Tween 20, 1%
BSA). The plates were washed three times with wash
buffer, and 100 ml of standard, control, or sample
blood was added to duplicate wells for overnight
incubation at 48C. The plates were washed three times
with wash buffer and incubated for 1 h at room
temperature with TGFb mAb (Genzyme MA, USA)
1 mg/ml in ELISA buffer. The plates were then washed
three times with ELISA wash buffer and incubated an
additional hour with biotinylated anti-mouse IgG
(Vector, Burlingame, CA, USA) 1 : 2000. Avidin-perox-
idase complex and the substrate were then added.
Orthophenylene diamine (Sigma Chem. USA) pre-
pared at 5 mg/ml, and 0.05 M hydrogen peroxide was
added and left 30 min at room temperature and the
plates read at 492 nm.
For quanti®cation of TNFa, IFNg and IL10 three
different kinds of human cytokine-speci®c mAb were
used (PharMingen, San Diego, CA, USA). Brie¯y, the
wells of 96-well microtiter plates (Immulon I, Nunc,
Roskilde, Denmark) were coated with 1 ± 2 mg/ml of the
capture mAb of each cytokine in 0.1 M NaHCO3
(pH=8.5) and incubated overnight at 48C. Following
blocking with 3% dry milk in PBS at room temperature
for 2 h, samples and standard recombinant IFNg, TNFa
and IL-10 were added and incubated overnight at 48C.
Then, 0.5 ± 2.0 mg/ml of biotinylated mAb for the
detection of human IFNg, TNFa and IL10 were added
followed by 1/400 avidin-peroxidase (Sigma Chem.
USA). Finally, the peroxidase substrate and the stop
solution were used to obtain OD determined at 492 nm.
Materials and methods
Patients
Three groups of patients were studied: those with
active MS, those with MS in remission and normal
controls. Patients were evaluated in this study
according to the criteria of Poser et al,16 with patients
free of clinical manifestation for more than 6 months
being considered to have a stable form of the disease
(remission) or with active disease in the last 6 months
(relapses). Sixty-three individuals were studied: 31 (26
women and 5 men) with stable MS; 15 (11 women and
4 men) with active MS and 17 (13 women and 4 men)
normal subjects. The EDSS was 2.6+1.2 (mean+s.d.).
The individuals had a mean age of 31+2 years and all
subjects came from the same geographic region and
same mixed ethnic origin. With the exception of two
subjects (who had received immunosuppressive drugs
and corticosteroid treatment 2 months prior to the
blood donation), none of the subjects had received any
treatment prior to donating blood for the study.
Antigen, antibody and recombinant cytokines
Polyclonal chicken anti-TGFb1 antibody was pur-
chased from R&D Systems (Minneapolis, MN, USA);
puri®ed bovine TGF-b1 and monoclonal mouse anti-
TGFb1 were provided by Genzyme, MA, USA; and
anti mouse IgG peroxidase was also obtained (Vector
Burlingame, CA, USA). The following reagents were
purchased from PharMingen (San Diego, CA, USA), IL-
10 (clones JES3-9D7; JES3-12G8) mAb, IFN-g (clones
NIB42;4S.B3) mAb, TNFa (clones Mab1;Mab11) mAb
and recombinant TNFa, IL-10 and IFN. Human Myelin
Basic Protein was obtained according to Deibler et al.17
Statistical analysis
Results are expressed as the mean+1s.d. of the mean,
and statistical signi®cance (P50.05) was analyzed by
the Mann-Whitney U-test.
Results
TNFa production from peripheral blood cells
Whole blood cells of patients with active and stable
MS and healthy controls were stimulated with LPS
and PHA in vitro, and the TNFa in the plasma was
quanti®ed using an ELISA assay. The TNFa was
measured in samples of blood from 31 patients with
clinically stable MS, 15 with the active disease, and 17
healthy control subjects. When the cells were stimu-
lated with LPS, no signi®cant differences between
means of TNFa were observed for the stable and
healthy controls (243+201 pg/ml versus 132+154 pg/
ml respectively; P40.05; patients with active MS
produce signi®cantly more TNFa than the healthy
controls (355+368 pg/ml versus 132+154 pg/ml re-
spectively; P50.05 (Figure 1a). When the cells were
stimulated in vitro with PHA, the mean TNFa was
356+418 pg/ml and 180+200 pg/ml for stable and
healthy controls, with 162+98 and 98+72, without
PHA, with no signi®cant difference found (P40.05).
Determination of cytokines synthesis
Lymphocytes were cultured in 2 ml RPMI 1640
medium supplemented with 2% FCS, at a cell density
of 26106 ml. The cells were then stimulated with
either MBP (25 mg/ml) or PHA (10 mg/l): 40 h for IL-10
production, 60 h for IFNg and 72 h for TGFb. For TNFa
production, the whole blood cells were stimulated
with either LPS (100 ng/ml) for 2 h or PHA (10 mg/ml)
for 24 h.
Capture ELISA for quantitation of cytokines
TGFb was measured using a capture ELISA developed
in the laboratory.18 TGFb Antibody (polyclonal anti-
body obtained from R&D, MN, USA) (1 mg/ml in PBS,
pH 7.4) was added to 96 well microtiter plates
(Immulon I, Nunc, Roskilde, Denmark). After overnight
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Changes in patterns of cytokines in relapsing and remitting MS
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295
For patients with active disease, the mean was
433+470 pg/ml stimulated with PHA and
128+64 pg/ml without PHA, again with no signi®cant
difference from the control (P50.05) (Figure 1b).
supernatants was found for patients with an active
form of the disease and the healthy controls. For
patients with stable disease the mean was
(702+607 pg/ml
stimulated
with
MBP
and
634+465 pg/ml without MBP versus 642+668 pg/ml
and 489+390 pg/ml, respectively; P40.05 (Figure 2b).
IFNg production from peripheral blood cell
Mononuclear cells from patients with active and stable
MS and healthy controls were stimulated for 60 h with
PHA (10 mg/ml) and MBP (25 mg/ml) and IFNg
production was quanti®ed by a capture ELISA assay.
For this experiment, 11 patients with active MS, 15
with stabilized MS, and 12 healthy controls were used.
When the cells were stimulated in vitro with PHA, the
IL-10 production by peripheral blood cells
Mononuclear cells from patients with active and stable
MS and healthy controls were stimulated for 40 h with
PHA or MBP, and IL-10 was quanti®ed by a capture
ELISA assay. For this part of the study, eight patients
with active MS, 15 in remission, and 17 healthy
controls were used. When the cells were stimulated by
PHA in vitro, the mean IL-10 production was
mean
IFNg
was
1205+1092 pg/ml
and
1315+1060 pg/ml for stable and healthy controls,
with 182+128 pg/ml and 498+390 pg/ml, without
PHA, with no signi®cant difference found (P40.05).
For patients with active disease, the mean was
1725+720 pg/ml
versus
1701+950 pg/ml
and
469+398 pg/ml versus 402+396 pg/ml without PHA
for stable patients and healthy controls. There was no
signi®cant difference between the two groups
(P40.05). Mean IL-10 production by mononuclear
cells from patients with active disease however, was
548+419 when stimulated with PHA (398+324 with-
out stimulation), representing a signi®cant decrease of
the production of IL-10 in relation to healthy controls
(P=0.003) (Figure 3a).
2393+2148 pg/ml
stimulated
with
PHA
and
634+465 pg/ml without PHA, again with no signi®-
cant difference from the control (P40.05) (Figure 2a).
Stimulation with MBP resulted in a somewhat
lowered IFNg production for the stable patients in
relation to the healthy controls (223+430 pg/ml
stimulated with MBP and 182+128 without MBP
versus 642+668 pg/ml and 489+390 pg/ml) respec-
tively P50.03; no differences in presence in the
In vitro MBP stimulation of mononuclear cells led to
signi®cant increase in mean IL-10 production
a
Figure 1 Production of TNFa by MS patients with active ~
or stable disease & and healthy controls !. After whole
peripheral blood cell stimulation with LPS (100 ng/ml) for 2h
(a) and PHA for 24 h (10 mg/ml) (b), TNFa concentration
were detected by ELISA assay. Results corresponding to
results from stimulated cells minus results for unstimulated
cells. *(P50.03)
Figure 2 Production of IFNg by MS patients with active ~
or stable disease and healthy controls !. After
lymphocyte stimulation in vitro with or PHA (10 mg/ml) (a)
or MBP (25 mg/ml), (b) IFNg were detected by ELISA in
supernatants from 60 h culture. Results corresponding to
results from stimulated cells. (P50.05)
&
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Changes in patterns of cytokines in relapsing and remitting MS
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296
1430+627 pg/ml when stimulated with MBP and
469+398 without MBP versus 385+254 pg/ml and
402+396 for stable MS and healthy controls, respec-
tively; (*P50.01). No signi®cant differences in the
mononuclear cells subjected to in vitro MBP activation
from active and the healthy controls were observed
(482+350 pg/ml
stimulated
with
MBP
and
398+324 ng/ml without MBP versus 385+254 and
402+396 pg/ml, without MBP) respectively; (P40.05)
(Figure 3b).
Production of TGFb from mononuclear cells
Blood from seven patients with active MS, 15 with
stable MS and 15 healthy controls were studied. In
vitro PHA and MBP stimulation of cells for 72 h was
followed by TGFb quanti®cation. The mean TGFb
production by mononuclear cells from patients with a
stable form of the disease stimulated with PHA was
921+1067 pg/ml (632+543 pg/ml without PHA) ver-
sus 983+967 pg/ml (253+198 pg/ml without PHA),
for the healthy control group. The production in the
active disease group 699+487 pg/ml (296+298 pg/ml
without PHA) versus 983+967 pg/ml (253+198 pg/ml
without PHA) was somewhat lower, although the
difference did not reach signi®cant levels in statistical
terms (P40.05) (Figure 4a).
Figure 4 Production of TGFb by MS patients with active ~
or stable disease and healthy controls !. After
lymphocyte stimulation with PHA (10 mg/ml (a) or MBP
(25 mg/ml (b), TGFb were detected by ELISA in supernatants
from 72 h culture. Results corresponding to results from
stimulated cells. *(P50.05)
&
Peripheral blood cells from stable patients stimulated
with MBP produced signi®cantly greater amounts of
TGFb (1700+1461 pg/ml stimulated and 632+543 pg/
ml (unstimulated) versus 254+264 pg/ml (stimulated)
and 253+198 pg/ml (unstimulated) (*P50.002) for
stabilized and healthy controls, respectively. No sig-
ni®cant differences in mononuclear cells subjected to in
vitro MBP activation from active and the healthy
controls were observed (201+169 pg/ml (296+298
without MBP) versus 254+264 pg/ml (253+198 pg/ml
without MBP) respectively; P40.05 (Figure 4b).
Discussion
These data show that changes in cytokine secretion
patterns do occur in the peripheral blood cells of
patients with active and stable MS. Whole leukocytes
were used because, although Th1 and Th2 cells are the
major sources of their respective cytokines, many other
cells both within and outside of the immune system
also produce such cytokines. Since various cell types
can contribute to the overall Th1 and Th2 cytokine
pattern, it has been suggested that these responses
should possibly be described as type 1 and type 2 cell
response. For example: subsets of gd T cells, CD4 and
CD8 can secrete Th1 and Th2-like cytokine patterns.
NK cells also produce IFNg and TNFa and contribute
to Th1-like responses,19 moreover, IL-4 and possibly
other Th2 cytokines are synthesized by mast cells, B
cells, basophils and NK 1.1 cells.20 Furthermore, IL-10
is produced by macrophages and B cells.21
Figure 3 Production of IL-10 by MS patients with active ~
or stable disease
& and healthy controls !. After
lymphocyte stimulation with PHA (10 mg/ml (a) or MBP
(25 mg/ml) (b), IL-10 levels were detected by ELISA in
supernatants from 40 h culture. Results corresponding to
results from stimulated cells. *(P50.05)
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Changes in patterns of cytokines in relapsing and remitting MS
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Although many interesting type
1
or type
2
of EAE in murines. Further support for this protective
role of IFNg during the pathogenesis of EAE has come
from studies showing that mice with disrupted IFNg
gene are susceptible to the induction of the disease.29
The characteristic cytokine products of Th1 and
Th2 are mutually inhibitory for the differentiation and
effector function of the reciprocal phenotype. IFNg has
been shown to prevent Th2 cell proliferation, whereas
IL-10 profoundly inhibits the synthesis of Th1
cytokines.30 ± 32 TGFb has been described as a Th3
cytokine33 and its effects on suppressing EAE have also
been demonstrated.10 The present study has shown
that in the active phase of the disease there is a
decrease in the level of IL-10 when cells are stimulated
with PHA, whereas no differences in TGFb levels were
found, despite previous suggestions to the contrary
published in the literature. Patients with stable forms
of the disease revealed no differences in the produc-
tion of IL-10 and TGFb when stimulated under similar
conditions. Patients with stable forms of the disease,
however, were found to reveal increases in the
production of IL-10 and TGFb when stimulated with
MBP.34
responses have been observed in autoimmune dis-
eases, a careful consideration of anatomical location
and the kinetics of a response can provide more
de®nitive data. Moreover, many immune responses
may remain localized so that sampling of peripheral
blood lymphocytes may provide information about
potential responses, whereas sampling of CSF or brain
tissue represents an ongoing response. Peripheral
blood was selected for investigation of the cytokines
patterns here since MS involves both a localized
immune response in the CNS and various immune
abnormalities correlated with disease activity in the
peripheral immune compartment.22
Proin¯ammatory cytokines such as TNFa and IFNg
are associated with disease activity in both MS
patients and the experimental model of Experimental
Autoimmune Encephalomyelitis (EAE).6,7 Since TNFa
is a major inducer of endothelial adhesion molecules
and chemokines, it can be predicted that it will have a
major effect on the recruitment of lymphocytes by the
central nervous system.23 A recent study describes the
spontaneous development of a chronic in¯ammatory
demyelinating disease in transgenic mice that consti-
Myelin basic protein seems to have stimulated the
production of anti-in¯ammatory cytokines in the cells
from patients with stable MS; the production of anti-
in¯ammatory cytokines by T cell clones from MS
patients sensitized to PLP has also been observed34,35
The mechanisms for stimulation of the increased
production of anti-in¯ammatory cytokines while in-
hibiting that of pro-in¯ammatory ones by neuroanti-
gens such as MBP are not well understood. In the
Lewis rat model, administration of myelin basic
protein has been shown to be an effective means of
suppressing EAE, an effect mediated by the TGFb,
secreted by T-cells from orally tolerized animals after
being triggered by the oral tolerogen. We previously
demonstrated that suppressor determinants exist on
the MBP molecule, since T cells from animals fed with
21 ± 40 peptide secreted TGFb, whereas no TGFb was
released in animals fed with MBP encephalitogenic
determinant (71 ± 90).36 It is possible that suppressive
determinants present on the MBP molecule stimulate
the production of anti-in¯ammatory cytokines (IL-10
and TGFb) by MBP-speci®c T cells from patients with
stable MS. The increased levels of anti-in¯ammatory
cytokines, on the other hand, may inhibit the
production of pro-in¯ammatory cytokines such as IFNg,
as seen in this study.
tutively have
a dysregulate murine TNFa gene.
Transgenic expression was restricted to the central
nervous system, and the direct involvement of TNFa
in the pathogenesis of the disease was con®rmed by
peripheral administration of neutralizing murine TNFa
antibodies.24 The present study showed that in vitro
LPS stimulation of peripheral blood cells from patients
with active MS led to a moderate increase in TNFa
production; this is in agreement with several authors
who revealed an increase in TNFa in patients with
active MS.23,24 The stimulation of lymphocytes using
PHA, however, did not induce a signi®cant difference
in TNFa production for the individuals in the groups
studied.
No increase in IFNg production in the active form of
the disease was induced by MBP stimulation nor by
polyclonal activation with PHA. These results are in
contrast to previous studies,25,26 where the authors
found an increase in the levels of this cytokine. On the
other hand, the data found here are in agreement with
previous observations which found no signi®cant
increase in IFNg levels during the active phase of
disease. There was, indeed, a signi®cant reduction in
level of IFNg observed in patients with a stable form of
the disease when the cells were stimulated with MBP.
Until recently, observations pointed to a critical role
for IFNg in the pathogenesis of MS and in the EAE
model, although recent data using a variety of systemic
and CNS-localized manipulations of IFNg production
have led to the questioning of the overall importance
of this cytokine in disease initiation and progression.
A number of investigations have shown that systemic
administration of neutralizing IFNg antibodies leads to
exacerbation of the disease in both susceptible and
resistant strains of mice, rather than the amelioration
of clinical signs.27,28 In addition, the in vivo adminis-
tration of IFNg has been found to decrease the severity
The results of stimulation of the cells of patients
with the active form of the disease suggest a reduction
in clones producing anti-in¯ammatory cytokines in the
peripheral blood since even stimulation with poly-
clonal mitogens such as PHA was not capable of
simulating the production of signi®cant levels of these
cytokines.
Thus, in autoimmune diseases, type
1 and 2
cytokines may polarize forms of the speci®c immune
response that result from the combined action of
genetic and environmental conditions. Consequently,
inhibiting mechanisms such as the production of anti-
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Changes in patterns of cytokines in relapsing and remitting MS
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298
14 Rott O, Fleisher B, Cash B. (1994) Interleukin-10 prevents
experimental allergic encephalomyelitis in rats. Eur J
Immunol 24: 1434 ± 1440.
in¯ammatory cytokines can be used to interrupt the
progression of events leading to chronic in¯ammation
thus, seems to be a promising way of treating organ-
speci®c autoimmune diseases such as MS.
15 Khoury SJ, Hancock WW, Weiner HL. (1992) Oral
tolerance to myelin basic protein and natural recovery
from experimental autoimmune encephalomyelitis are
associated with down-regulation of in¯ammatory cyto-
kines and differential upregulation of transforming
growths factor b, interleukin-4 and prostaglandin E
expression in the brain. J Exp Med 176: 1355 ± 1364.
16 Poser CM et al. (1983) New diagnostic criteria for
multiple sclerosis: guidelines for research protocols.
Ann Neurol 13: 227 ± 231.
Acknowledgements
The authors would like to acknowledge the support
of FAPESP (Grant #94/5707-2; 96/2044-8); (CNPq
522282/94-96) and FAEP-UNICAMP. They would
also like to acknowledge the collaboration of Linda
Gentry EL-Dash in the linguistic revision of the
manuscript.
17 Deibler GE, Martenson RE, Kies MW. (1972) Large scale
preparation of myelin basic protein from central nervous
tissue of several mammalian species. Prep Bioch 2: 139 ±
165.
18 Santos LMB et al. (1994) Oral Tolerance to myelin basic
protein induces regulatory TGFb secreting T cells in
Peyer's patches. Cell Immunol 157: 439 ± 447.
19 Mosmann TR, Sad S. (1996) The expanding universe of
T-cell subsets: Th1, Th2 and more. Immunology Today
138: 138 ± 146.
References
1 Waksman B. (1985) Mechanisms in multiple sclerosis.
Nature 318: 104 ± 105.
2 Ota K et al. (1990) T cell recognition of an immunodo-
minant myelin basic protein epitope in multiple sclero-
sis. Nature 346: 183 ± 187.
20 Seder RA, Paul WE. (1994) Acquisition of lymphokine-
producing phenotypes by CD4+
T cells. Ann Rev
Immunol 12: 635 ± 673.
3 Sharief MK, Hentges R. (1991) Association between
tumor necrosis factor a and disease progression in
patients with Multiple sclerosis. N Engl J Med 325:
467 ± 472.
21 D'Orazio TJ, Niederkon JY. (1998) A novel role of TGFb
and IL 10 in the induction of immune privilege. J
Immunol 160: 2089 ± 2098.
22 Ha¯er DA, Weiner HL. (1989) Multiple sclerosis a CNS
and systemic autoimmune disease. Immunology Today
10: 104 ± 107.
23 Barten DM, Ruddle NH. (1994) Vascular cell adhesion
molecule-1 modulation by tumor necrosis factor in
multiple sclerosis. J Neuroimmunol 51: 123 ± 133.
4 Hartung HP et al. (1995) Circulating adhesion molecules
and tumor necrosis factor in multiple sclerosis: correla-
tion with magnetic resonance imaging. Ann Neurol 38:
186 ± 193.
5 Choffon M, Jillard C, Gauthier C, Grau G. (1992) Tumor
necrosis factor production as possible predictor of
relapse in patients with multiple sclerosis. Eur Cytokine
Netw 433: 523 ± 531.
6 Panitch HS, Hirsch RL, Schindler J, Jonhnson KP. (1987)
Treatment of multiple sclerosis with gamma interferon:
exacerbations associated with activation of the immune
system. Neurology 37: 1097 ± 1102.
7 Navikas V, Link H. (1996) Review. Cytokines and the
pathogenesis of multiple sclerosis. J Neurosc Res 45:
322 ± 333.
8 Rieckmann P et al. (1994) Tumor necrosis factor a
messenger RNA expression in patients with relapsing-
remitting multiple sclerosis is associated with disease
activity. Ann Neurol 37: 227 ± 257.
9 Van der Veen RC, Stohlman SA. (1993) Encephalitogenic
Th1 cells are inhibited by Th2 cells with related peptides
speci®city: relative roles of IL-4 and IL-10. J Neuroim-
munol 48: 213 ± 220.
10 Schluesener HJ, Lider O. (1989) Transforming growth
factor b1 and b2 cytokines with identical immunosup-
pressive effects and a potential role in the regulation of
autoimmune T cell function. J Neuroimmunol 24: 249 ±
258.
11 De Waals Malefyt R et al. (1991) Interleukin-10 inhibits
cytokines synthesis by human monocytes: an autoregu-
latory role of IL-10 produced by monocytes. J Exp Med
174: 1209 ± 1220.
24 Probert
L et al. (1995) Spontaneous in¯ammatory
demyelinating disease in transgenic mice showing
central nervous system-speci®c expression of tumor
necrosis factor alpha. Proc Natl Acad Sci USA 92:
11294 ± 11298.
25 Brod SA et al. (1995) Decreased CD3 mediated interferon
g production in relapsing-remitting multiple sclerosis.
Ann Neurol 37: 546 ± 549.
26 Link J et al. (1994) Organ-speci®c autoantigens induce
interferon gamma and interleukin 4 mRNA expression in
mononuclear cells in multiple sclerosis and myasthenia
gravis. Neurology 44: 728 ± 734.
27 Willenborg DO et al. (1996) IFN-gamma plays a critical
down-regulatory role in the induction and effector phase
of myelin oligodendrocyte glycoprotein-induced auto-
immune encephalomyelitis. J Immunol 157: 3223 ± 3227.
28 Haremans H et al. (1996) Chronic relapsing experimental
autoimmune encephalomyelitis (CREAE) in mice: en-
hancement by monoclonal antibodies against interferon-
gamma. Eur J Immunol 26: 2393 ± 2398.
29 Ferber IA et al. (1996) Mice with a disrupted IFNg gene
are susceptible to the induction of experimental auto-
immune encephalomyelitis (EAE). J Immunol 156: 5 ± 7.
30 Kennedy MK, Torrence DS, Pichia KS, Mohler KM.
(1992) Analysis of cytokine mRNA expression in the
central nervous system of mice with experimental
autoimmune encephalomyelitis reveals that IL-10 mRNA
expression correlates with recovery. J Immunol 149:
2496 ± 2505.
12 Nicholson LB, Kuchroo VK. (1996) Manipulation of the
Th1/Th2 balance in autoimmune diseases. Curr Opin
Immunol 8: 837 ± 842.
31 Musette P et al. (1996) The pattern of reproduction of
cytokine mRNA is markedly altered at the onset of
Multiple Sclerosis. Res Immunol 147: 435 ± 441.
13 Liblau RS, Singer SM, McDevitt HO. (1995) Th1 and Th2
CD4+ T cells in the pathogenesis of organ-speci®c
autoimmune disease. Immunol Today 16: 34 ± 38.
Multiple Sclerosis
Downloaded from msj.sagepub.com at SETON HALL UNIV on September 16, 2014

Changes in patterns of cytokines in relapsing and remitting MS
P Mara da Costa et al
299
32 Rieckmann P et al. (1994) Cytokine mRNA levels in
mononuclear blood cells from patients with active
Multiple Sclerosis. Neurology 44: 1523 ± 1526.
33 Chen Y et al. (1994) Regulatory T cell clones induced by
oral tolerance: suppression of experimental autoimmune
encephalomyelitis. Science 265: 1237 ± 1240.
34 Mokhtarian F et al. (1994) Defective production of anti-
in¯ammatory cytokine TGFb by T cell lines of patients
with active multiple sclerosis. J Immunol 152: 6003 ±
6011.
35 Correale J et al. (1995) Patterns of cytokine secretion by
autoreactive proteolipid protein speci®c T cell clones
during the course of multiple sclerosis. J Immunol 154:
2959 ± 2968.
36 Miller A et al. (1993) Epitopes of myelin basic protein
that trigger TGF b release after oral tolerization are
distinct from encephalitogenic epitopes and mediate
epitope-driven bystander suppression. J Immunol 151:
7307 ± 7315.
Multiple Sclerosis