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Elevated osteopontin levels in active relapsing-remitting multiple sclerosis.

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Elevated Osteopontin Levels in
Active Relapsing-Remitting
Multiple Sclerosis
Mario H. J. Vogt, PhD,1 Luba Lopatinskaya, MD,1,2
Monique Smits, BSc,1 Chris H. Polman, MD, PhD,2
and Lex Nagelkerken, PhD1
In the search for proteins that might play a role in the
pathogenesis of multiple sclerosis (MS), osteopontin
(OPN) has been identified as the most prominent
cytokine-encoding gene expressed within MS lesions. Here,
we report significantly increased OPN protein levels in
plasma of relapsing-remitting MS patients. In contrast,
OPN protein levels in primary progressive and secondary
progressive MS patients were similar to healthy control
levels. Interestingly, active relapsing-remitting patients had
higher OPN protein levels than patients without relapses.
Ann Neurol 2003;53:819 – 822
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system.1,2 High-throughput
sequencing of cDNA from MS brain lesions and control brains has showed that osteopontin (OPN) is the
most abundant cytokine-encoding transcript unique to
MS plaques.3 MS tissues have been probed immunohistochemically for the expression of OPN, and its
level was particularly elevated in reactive astrocytes and
microglial cells.3
OPN is a negatively charged acidic hydrophilic protein of 314 amino acids and has pleiotropic functions,
including a role in both acute and chronic inflammation.4 OPN is involved in the recruitment of macrophages and T cells to inflamed sites.5,6 An interaction
between the C-terminal domain of OPN and the receptor CD44 expressed by macrophages induces chemotaxis, and engagement of ␤3-integrin receptors by
the N-terminal OPN domain induces activation.7 Interestingly, mice deficient in OPN gene expression
(OPN⫺/⫺) have severely impaired type 1 immunity to
viral and bacterial infections.6 OPN⫺/⫺ mice were also
resistant to progressive EAE and had frequent remis-
From the 1Division of Immunological and Infectious Diseases,
TNO Prevention and Health, Leiden; and 2Department of Neurology, VU Medical Center, Amsterdam, The Netherlands.
Received Feb 7, 2003, and in revised form Mar 7. Accepted for
publication Mar 18, 2003.
Address correspondence to Dr Vogt, Division of Immunological and
Infectious Diseases, TNO Prevention and Health, P.O. Box 2215,
2301 CE, Leiden, The Netherlands. E-mail: mhj.vogt@pg.tno.nl
sions, indicating that OPN plays an important role in
the demyelination process.3,8 Cells from draining
lymph nodes from OPN⫺/⫺ mice demonstrated impaired interleukin (IL)–12 and IFN␥ production and
increased IL-10 production compared with OPN⫹/⫹
mice, suggesting that OPN is a T helper cell 1 (Th1)
cytokine.
In view of the functional role of OPN and its ubiquitous tissue expression, we determined whether OPN
protein levels in serum of MS patients were associated
with disease activity.
Subjects and Methods
Patients and Controls
Thirty relapsing-remitting (RR), 10 secondary progressive
(SP), and 10 primary progressive (PP) MS patients at the
Department of Neurology, VU Medical Center, Amsterdam
were included in this study for determination of OPN protein baseline levels. Ten healthy volunteers were included as
a control group. The demographic characteristics of the MS
patients are given in the Table. Relapse was defined as the
appearance or reappearance of one or more neurological abnormalities that persisted for at least 24 hours, and which
had been preceded by at least 30 days of stable or improved
neurological state. Twenty of these RR MS patients were selected for a prospective immunological and clinical follow-up
over the course of 6 months immediately after initiation of
interferon (IFN)–␤1b treatment. Treatment consisted of
subcutaneous injections of 8 million international units of
IFN-␤1b on alternate days. EDTA blood was collected at 0
(baseline) and 1, 3, and 6 months after initiation of the therapy.
Enzyme-linked Immunosorbent Assay
The OPN content in EDTA plasma was measured using a
standard ELISA according to the manufacturer’s procedure
(Assay Designs, Ann Arbor, MI).
Data Analysis
All statistical tests were performed using the MS Windows
SPSS 11.0 package.
One-way analysis of variance followed by a post hoc analysis (Bonferroni) was used for group comparisons in the
cross-sectional approach. For all serial samples, the analysis of
variance module (univariate general linear model) was used.
In the model, the subjects were taken as a random factor,
and different time points were included as fixed factors.
Once a significant difference among the time groups was
found, the Bonferroni post hoc test was used to determine
which pairs of groups differed. Reported p values were two
tailed and p value less than 0.05 was considered significant in
this exploratory study.
Results
Baseline Osteopontin Protein Levels in Multiple
Sclerosis Patients and Controls
OPN protein levels in serum of MS patients with different clinical subtypes were evaluated and compared
© 2003 Wiley-Liss, Inc.
819
Table. Demographic and Clinical Characteristics of Multiple Sclerosis Patients
Clinical Subtypes
No. of patients
Gender (female to male ratio)
Mean age (yr ⫾ SD)
Disease duration (yr ⫾ SD)
EDSS at intake (mean ⫾ SD)
PP
RR
SP
10
6:4
51.2 ⫾ 12.6
14.0 ⫾ 9.8
6.1 ⫾ 1.6
30
25:5
39 ⫾ 6.2
7.8 ⫾ 5.3
3.6 ⫾ 1.5
10
5:5
47.4 ⫾ 6.9
12.2 ⫾ 7.7
4.9 ⫾ 1.1
PP ⫽ primary progressive; RR ⫽ relapsing remitting; SP ⫽ secondary progressive; SD ⫽ standard deviation. EDSS ⫽ expanded disability
status scale.
with levels present in healthy controls. As depicted in
Figure 1, the median OPN concentration in 10 healthy
controls was 175ng/ml, and the levels showed only little variation between subjects. PP MS patients had a
slightly increased median value of 224ng/ml, demonstrating high variation of OPN levels between subjects.
However, the OPN levels in the PP MS group did not
significantly differ from levels in healthy controls. In
contrast with the PP MS group, OPN levels in the RR
MS group were significantly ( p ⬍ 0.001) enhanced by
60% compared with the levels in healthy controls.
OPN levels in SP patients were comparable to levels in
healthy controls.
Clinical Exacerbations and Osteopontin Protein
Levels in Plasma of Relapsing-remitting Multiple
Sclerosis Patients
The OPN protein levels in the RR MS patient group
displayed a rather large variation between subjects.
Because these patients develop repeated clinical exacerbations followed by remissions, the variance of
Fig 1. Osteopontin (OPN) protein levels in plasma of healthy
controls (HCs), primary progressive (PP), relapsing-remitting
(RR), and secondary progressive (SP) multiple sclerosis (MS)
patients. OPN protein concentration in EDTA plasma of each
subject was determined using a standard enzyme-linked immunosorbent assay. The line represents the median value of each
patient group. RR MS patients had significantly elevated OPN
protein levels compared with the healthy controls (Bonferroni’s
post hoc after one-way analysis of variance, p ⬍ 0.001).
820
Annals of Neurology
Vol 53
No 6
June 2003
OPN levels may be the result of differences in disease
activity at the time of sampling. Out of the 30 abovementioned RR patients, 20 received treatment with
IFN-␤1b, and their disease activity was very well documented both before and after start of treatment. A
total of 13 relapses were noticed in 11 of these patients during the time period from 3 months before
until 3 months after sampling of plasma for OPN
protein baseline evaluation. OPN protein baseline
levels were significantly ( p ⫽ 0.019) enhanced in
these 11 patients who developed relapses as compared
with the nine patients in which no relapse was noticed during this interval (Fig 2). In fact, OPN levels
in the latter group were not significantly ( p ⫽ 0.218)
different from the levels observed in the healthy control group.
Fig 2. Elevated osteopontin (OPN) protein levels in plasma of
relapsing-remitting multiple sclerosis patients who developed
relapses. OPN protein concentrations in EDTA plasma of each
subject was determined using a standard enzyme-linked immunosorbent assay. During the time period of 3 months before
until 3 months after sampling, 13 relapses were noticed in 11
of 20 patients. Those patients who developed relapses had significantly (p ⫽ 0.019) higher OPN protein baseline levels
compared with the nine patients for whom no relapse was
noticed during this observation period. The line represents the
median value of each patient subgroup.
Effect of Interferon-␤1b Treatment on Osteopontin
Protein Levels in Relapsing-remitting Multiple
Sclerosis Patients
To determine whether IFN-␤1b treatment had an effect on the OPN protein levels in the RR MS patients, the OPN plasma concentration in each patient
was measured before initiation of IFN-␤1b treatment
and compared with the levels after 1, 3, and 6
months of treatment. The effect of IFN-␤1b on OPN
protein levels is depicted as percentage difference
from OPN baseline levels (Fig 3). Surprisingly, OPN
protein levels were slightly but significantly ( p ⬍
0.001) enhanced after 1 month of IFN-␤1b treatment, with a 15% increase in median value compared
with baseline samples (T0). However, after 3 and 6
months of IFN-␤1b therapy OPN levels declined to
baseline levels again.
Discussion
In the search for immunomodulatory proteins that
are important in MS, microarray analysis and highthroughput sequencing of cDNAs from MS brain lesions showed that the level of transcripts for OPN
was elevated.3 Because OPN is a ubiquitously expressed protein, OPN upregulation in various cell
types may result in elevated plasma levels of this protein in MS patients. In this study, we demonstrated
that RR MS patients had significantly increased OPN
levels, whereas PP and SP MS patients had similar
OPN levels as healthy controls. The OPN protein
levels in the RR MS patient group displayed a rather
large variation between subjects. We showed that the
Fig 3. Effect of recombinant interferon (IFN)–␤1b treatment
on osteopontin (OPN) protein levels in plasma of relapsingremitting MS patients. OPN protein concentrations in EDTA
plasma of each subject was determined using a standard
ELISA. The OPN protein level in each subject was measured
at initiation of IFN-␤1b treatment (OPN T0) and after 1
(OPN T1), 3 (OPN T3) and 6 months (OPN T6) of treatment. The percentage differences were calculated as (OPN
Tx ⫺ OPN T0)/OPN T0*100. OPN protein levels were
slightly but significantly upregulated during the first month of
IFN-␤1b treatment (p ⬍ 0.001), followed by a decline to
baseline from 3 months onward.
variance of OPN levels was the result of differences in
disease activity in each subject at the time of sampling. Patients with high disease activity had higher
OPN protein levels than patients without clinical exacerbations.
OPN has pleiotropic functions, including a role as
Th1 cytokine with the ability to increase IFN-␥ and
IL-12 production and to decrease IL-10 release by immune cells.6 Previously, we demonstrated that peripheral blood mononuclear cells in MS patients have increased levels of IL-12p40 and decreased levels of
IL-10 mRNA as compared with controls.9 Elevated expression of the IL-12p40 subunit has been found in
the cerebrospinal fluid of RR patients with active
plaques, and MS patients had increased numbers of
IFN-␥–secreting cells in peripheral blood and cerebrospinal fluid compared with controls.10 These observations indicate that the Th1/Th2 balance is dysregulated. Increased levels of OPN in serum of RR MS
patients may contribute to this phenomenon.
IFN-␤ treatment of RR patients reduces relapse rate
by approximately 30%.11 The mechanism by which
IFN-␤ works remains unclear, although several possible
mechanisms have been suggested. IFN-␤ may inhibit
the entry of peripheral blood mononuclear cells into
the central nervous system by downregulation of adhesion molecules on brain endothelial cells and by antagonizing increased migration of peripheral blood mononuclear cells in response to chemokines.12,13 IFN-␤
may also have immunomodulatory effects on different
cell subsets and their cytokine production.14 For example, IFN-␤ treatment enhances IL-10 mRNA and protein levels and increases the percentage of IL10 –secreting peripheral blood T lymphocytes.15,16 Surprisingly, we found slightly but significantly enhanced
OPN protein levels in plasma of RR patients during
the first month of IFN-␤1b treatment. Studies indicate
that the OPN promoter contains various motifs including IFN-inducible elements.17,18 These elements may
be responsible for the initial OPN upregulation within
the first month of IFN-␤1b therapy. This finding is in
line with other studies, which showed that IFN-␤ is
able to induce Th1 markers.19 Moreover, it has been
demonstrated that IFN-␤1b treatment induces elevation of IFN-␥–producing cell numbers in MS patients
during the first 2 months of treatment, followed by a
normalization after 3 months.20 Similar effects of IFN␤1b on OPN protein levels have been observed in this
study.
In conclusion, increased OPN protein levels in
plasma of RR MS patients are associated with disease
activity. These increased levels may contribute to a dysregulation of the Th1/Th2 balance as has been suggested by several studies.
Vogt et al: Osteopontin Levels in MS
821
This work was supported by the Dutch Foundation for the Support
of MS Research (Stichting Vrienden MS Research).
We are grateful for Drs S. Dijkstra and H. van Noort for critically
reviewing the manuscript.
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