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Response to Comment on "The Influence of the Proinflammatory Cytokine, Osteopontin, on Autoimmune Demyelinating Disease"
Blom et al. (1) challenge
several studies (2-6) that showed that
osteopontin (OPN) playsa key role in the progression of autoimmune
disease in modelsof multiple sclerosis (MS) and rheumatoid arthritis.
Accordingto Blom et al., the results of the study by Chabas
et al. (2)and those reported in four other
papers (3-6)reflect a polymorphism
linked to OPN, rather than a role for theOPN protein itself. In this
response to their comment, we reviewthe results of the Chabas et
al. (2) study; offer newdata showing that vaccination
against OPN potently modulates experimentalautoimmune
encephalomyelitis (EAE), a model of MS; suggest somepossible reasons
for the difference between the Blom et al. (1)results and our own (2); and note some additional studiesthat reveal that OPN is involved in the pathophysiology not onlyof MS
but also of other neurological diseases (7-9).
Chabas et al. (2) reported that large-scale
sequencing of non-normalized cDNA libraries derived from plaques
dissectedfrom autopsies of three brains of individuals with MS
revealedthat OPN transcripts were among the most abundant. OPN
transcriptswere likewise elevated in gene microarray analysis of EAE
brainlesions. OPN was demonstrated in both MS and EAE lesions withimmunohistochemistry. Within active MS plaques, OPN was foundon
microvascular endothelial cells and macrophages and in whitematter
adjacent to plaques. Reactive astrocytes and microgliaalso expressed
OPN.
Progressive paralysis ensues in mice after injection of myelin
oligodendroglial glycoprotein (MOG) peptide 35-55 in completeFreund's
adjuvant (CFA). In OPN/ knockout mice maintained on a
129/C57BL/6 outbred strain (10),injected with MOG 35-55, progressive EAE is rare, whereas remissionsof disease are common. EAE
was observed in 100% of both OPN+/+ and
OPN/ mice with MOG 35-55, but the severity of disease
was reducedin all animals in the OPN/ group, and these
mice were totally protected from EAE-relateddeath (2).
During the first 26 days, OPN/ mice displayed a
distinct evolution of EAE, with a much higherpercentage of mice having
remissions compared to the controls.
Differences in cytokine expression in these mice confirmed that OPN was
pivotal in controlling Th1/Th2 polarization. T cellsin
OPN/ mice showed a reduced proliferative response to
MOG 35-55 comparedwith the response of OPN+/+ T cells. In
addition, IL-10 production was increased in T cellsreactive to MOG
35-55 in OPN/ mice that had developed EAE compared with
T cells in OPN+/+ mice. At the same time, IFN and
IL-12 production were diminishedin cultures of spleen cells stimulated
with MOG (2).Futhermore, we recently showed that
immunization with a DNA plasmid-encodingosteopontin
(11) alters the chronic course of EAE (Fig. 1A).DNA
immunization elicited antibodies to OPN, and disease modulationoccurred at a time when antibody titers to OPN were highest (Fig.
1B).
Fig. 1.
(A) Immunization with DNA encoding
OPN ameliorated MOG p35-55-induced EAE in C57BL/6 mice when
administered before EAE induction. Mice were immunized weekly,
intramuscularly for four weeks, with 100 µg of DNA vaccine encoding
OPN, vaccine without DNA encoding OPN, or PBS only (16).
Five days prior to first vaccination, mice were injected once with 10 µM cardiotoxin (Sigma). EAE was induced 1 week after the last
vaccination by immunization with 100 µg of MOG p35-55 emulsified in
CFA containing 4mg/ml heat-killed Mycobacterium tuberculosis
H37Ra (Difco Laboratories, Detroit, MI). On the day of immunization and
48 hours later mice were injected with 100 ng of Bordetella
pertussis toxin (BPT) in PBS, intravenously (i.v.). Mice were
examined daily for clinical signs of EAE and scored as follows: 0, no
paralysis; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb
paralysis; 4, hind limb and forelimb paralysis; and 5, moribund or
dead. Each group included 10 female mice 4 to 5 weeks old. Asterisk (*)
denotes p value <0.05, dagger () denotes
p value <0.001, in comparing OPN DNA vaccine group with
either of control groups. (B) Anti-OPN antibody production
was detected in sera following OPN DNA vaccination. At different time
points (0, 5, 14, 20, 30, and 40 days after EAE induction), sera were
collected and anti-self-OPN antibodies were measured. OPN antibodies
were detected after the DNA vaccination and boosted after EAE
induction. The specific antibody titer was detected using a direct
enzyme-linked immunosorbent assay (ELISA) (16). Results are
shown as mean log2 of five different samples. Asterisk (*)
denotes p value <0.002, dagger () denotes
p value <0.0001, in comparing OPN DNA vaccine group with
either of control groups.
[View Larger Version of this Image (32K GIF file)]
Jansson et al. (5) described concordant
results in another model of relapsing EAE--induced by proteolipid
proteinpeptide (PLP) p172-183--in C57BL/6X129 OPN/
knockout mice that had been backcrossed to C57BL/6 for six generations.Incidence of clinical disease and the mean day of onset were similarin
OPN/ and control, wild-type (OPN+/+)
littermates. The OPN/ animals, however, had lower mean
maximal clinical scores andfaster recovery without spontaneous
relapses; moreover, they displayeddecreased inflammatory infiltrates
and demyelination, and theirCD4+ T cells produced less
IFN and tumor necrosis factor-. Takentogether, these analyses
of the effect of targeted mutations ofOPN (2, 5)
in two different EAE models indicatethat OPN is likely to be a potent
modulator of autoimmune demyelinatingdisease.
The divergent results described by Blom et al.
(1) may be due to differences between the EAE model that
they studiedand the models used by our group (2) and by
Jansson etal. (5). Blom et al.
induced EAE with recombinant ratMOG 1-125, whereas we used a peptide
fragment of MOG 35-55, whichis the immunodominant epitope for B6.
Lyons et al. (12,13) showed that
"the form of inducing antigen (proteinversus peptide) plays
a role in the pathogenesis of EAE," (12)such that B
cell-deficient mice did not develop EAE when immunizedwith MOG,
although they were susceptible to MOG peptide 35-55-induceddisease.
Thus, different mechanisms are likely at work in EAEinduced with
recombinant proteins compared with the mechanismsin EAE induced with
peptide fragments of the same protein. Furthermore,the recombinant MOG
1-125 of rat origin, used by Holmdahl andcolleagues, has many
differences compared with the mouse sequence.
Finally, to definitively implicate linked genes as the basis for
differences between the data of Blom et al. (1)and those of our group (2) and Jansson et
al. (5),EAE induced in earlier backcrosses of the Blom
et al. OPN/ strain would have to be
examined. Until such differences areexplicitly analyzed, it is
difficult to draw any definitive conclusions.It is not at all unusual
for divergent results to appear in studiesof autoimmunity in knockout
mice, especially when different animalmodels are examined
(14).
Further recent studies, examining OPN polymorphisms and disease course
in MS, support a role for osteopontin in MS. In 821MS patients, a
trend for association with disease course was detectedin patients
carrying at least one1284A allele in the OPN gene.Patients
with this genotype were less likely to have a mild diseasecourse and
were at increased risk for a secondary-progressiveclinical type
(15). In addition, OPN transcripts wereelevated in
Huntington's disease, and transcripts and OPN proteinwere increased
in the brain in a transgenic mouse model of Huntington'sfollowing
successful treatment with cystamine (7). OPNtranscripts and
protein were also increased in an experimentalmodel of epilepsy
(8, 9).
In sum, the role of OPN in a wide variety of neuroinflammatory
and neurodegenerative conditions remains an area worthy ofintense
activity. The conflicting results from Blom et al.
(1)in the EAE model they used are of interest--but whether
the differencesare due to linked genes remains an open question at
present.
Lawrence Steinman Sawsan Youssef Natalie Van Venrooij Dorothée Chabas
Department of Neurological Sciences Stanford
University Stanford, CA 94305, USA Sergio E. Baranzini
Department of Neurology University of
California, San Francisco San Francisco, CA 94143, USA Susan Rittling
Department of
Genetics Rutgers University Piscataway, NJ 08854, USA David Denhardt
Department of Cell Biology and
Neuroscience Rutgers University Raymond A. Sobel
Department of Pathology Stanford University Christopher Lock Rosetta Pedotti
Department of Neurological Sciences Stanford University Jorge R. Oksenberg
Department of Neurology University of California, San Francisco
Ten mM cardiotoxin was given in the tibialis anterior muscle,
then 5 days later mice were given 100 µg of plasmid with full-length
OPN in the tibialis anterior muscle. The plasmid with OPN was injected
three more times at intervals of 6 to 7 days. EAE was induced 7 days
after the last injection with MOG 35-55, as described in
(2). Animals were scored as described in (2).
7 November 2002; accepted 16 February
2003
10.1126/science.1080223 Include this information when citing this paper.
The editors suggest the following Related Resources on Science sites:
In Science Magazine
TECHNICAL COMMENTS
Thomas Blom, Ahnders Franzén, Dick Heinegård, and Rikard Holmdahl (21 March 2003) Science299 (5614), 1845a.
[DOI: 10.1126/science.1078985] |Full Text »|PDF »
REPORTS
Dorothée Chabas, Sergio E. Baranzini, Dennis Mitchell, Claude C. A. Bernard, Susan R. Rittling, David T. Denhardt, Raymond A. Sobel, Christopher Lock, Marcela Karpuj, Rosetta Pedotti, Renu Heller, Jorge R. Oksenberg, and Lawrence Steinman (23 November 2001) Science294 (5547), 1731.
[DOI: 10.1126/science.1062960] |Abstract »|Full Text »|PDF »|Supplemental Data »
/
IFN and
IL-12 production were diminished in cultures of spleen cells stimulated
with MOG (
) denotes
p value <0.001, in comparing OPN DNA vaccine group with
either of control groups. (B) Anti-OPN antibody production
was detected in sera following OPN DNA vaccination. At different time
points (0, 5, 14, 20, 30, and 40 days after EAE induction), sera were
collected and anti-self-OPN antibodies were measured. OPN antibodies
were detected after the DNA vaccination and boosted after EAE
induction. The specific antibody titer was detected using a direct
enzyme-linked immunosorbent assay (ELISA) (
. Taken together, these analyses
of the effect of targeted mutations of OPN (
