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*
Neuroinflammation Group, Department of Neurochemistry, Institutes of Neurology and Ophthalmology, UCL,
Kennedy Institute of Rheumatology, Imperial College, and
Bone and Joint Research Unit, Queen Mary and Westfield College, University of London, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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) and
IFN-
(1). The spontaneous recovery of clinical disease
has been associated, in part, with increased production of mRNA coding
for the Th2/Th3 cytokines IL-10 and TGF-(2, 3). These
cytokines can inhibit the function of Th1 cells. The regulatory action
of TGF- in the recovery phase of EAE has been confirmed through
neutralization studies (4). However, the potential role of
IL-10 to regulate the disease process is more controversial.
IL-10 has been shown to be present in perivascular astrocytic endfeet
in the CNS of normal humans and MS patients (5). This
suggests an immunoregulatory role for IL-10 in the CNS and it has been
shown to inhibit EAE although one study showed human IL-10 to have no
effect or to exacerbate severity of EAE in mice (6, 7, 8, 9).
Interestingly, IL-10 has been reported to be elevated in the serum and
cerebrospinal fluid (CSF) of MS patients receiving IFN- therapy
(10). The role of IL-10 therapy in the CNS to inhibit EAE
has not been previously established. However, the short half-life of
biological agents such as cytokines requires their frequent
administration often in large doses to provide a therapeutic
concentration at the target organ. Therefore, the use of local gene
therapy in the CNS could be used to overcome the large daily systemic
administration of IL-10 needed for therapy of EAE. A previous study has
shown that genetically engineered syngeneic immortalized fibroblasts
are capable of long-term expression of therapeutic proteins in the CNS
with which to treat EAE (11). Using this approach, we have
demonstrated that immortalized fibroblasts infected with a retrovirus
coding for IL-10 can significantly ameliorate EAE. However, the nature
of the gene vector was found to be important, as adenoviral delivery of
IL-10 was found to be ineffective despite producing similar levels of
IL-10 protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Biozzi ABH mice were bred at the Institute of Ophthalmology. (University College London, London, U.K.) and fed rat-mouse expanded diet and water ad libitum. Mice aged between 6 and 8 wk old were injected s.c. in the flank with 1 mg mouse spinal cord homogenate (SCH) in IFA supplemented with 60 µg Mycobacterium tuberculosis H37Ra and Mycobacterium butyricum on days 0 and 7 as described previously (12). Animals were monitored daily and clinical signs scored as follows: 0, normal; 1, flaccid tail; 2, impaired righting reflex; 3, partial paralysis; 4, complete paralysis. Clinical signs of a lower severity than typically observed were scored 0.5 lower than the grade indicated as described previously (13).
Gene vectors
Mouse IL-10 cDNA was a kind gift from K. Moore (DNAX, Palo Alto, CA) and was cloned into the SalI site of pBabe-puro retrovirus containing a puromycin resistance gene, driven by Moloney murine leukemia virus long terminal repeats (14). The packaging cell line GPenv-AM12 was transfected with 20 µg Il10.pBabe-puro plasmid by the calcium phosphate precipitation method (15). Replication-deficient Il10.retroviral supernatant was collected and frozen at -70°C until used. Replication-deficient E1 deletion mutants of the type 5 human adenovirus coding for LacZ from Escherichia coli (AdRL) or mouse IL-10 (AdRIL10) driven by the Rous sarcoma virus promoter (a kind gift from M. J. Dallman, Imperial College, London, U.K.) were grown in 293 cells and purified twice by centrifugation on CsCl gradients, and the buffer was exchanged on a Sephadex column (16, 17).
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Immortalized fibroblast production and infection with retrovirus coding IL-10 |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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according to the manufacturer’s instructions (Promega,
Madison, WI). Controls consisted of tissue culture supernatant. |
Local administration and detection of IL-10 vectors in the CNS |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Isolation of immune infiltrate from the spinal cords of EAE mice |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Histology of spinal cord embedded in wax |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Immunohistochemistry |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Statistical analysis |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Results |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Previously it has been demonstrated that tsF cells can exist and
produce their transgene product for at least 5 wk following CNS
transplantation (11), whereas adenoviral transgenes are
expressed transiently, but at high levels (16). Samples
were collected 3 days post i.c. injection. CSF samples from 5 x
106 PFU ADRIL10 (n =
5) contained 110.3 ± 22.2 ng/ml IL-10 compared with mice injected
i.c. with 5 x 105 PFU AdRIL10
(n = 4), which contained 5 ± 1.6 ng/ml IL-10. CSF
from mice injected i.c. with 2 x 106
IL-10.tsF cells (n = 3) contained 19.0 ± 1.0
ng/ml IL-10. CSF samples from all other groups did not contain levels
of IL-10 above the threshold of detection by ELISA (15.0 pg/ml). Serum
IL-10 concentration from all groups was below the detection limit of
the ELISA (15.0 pg/ml). ELISA of tissue culture supernatants from
confluent immortalized mouse fibroblasts infected with retrovirus
coding for murine IL-10 (IL-10.tsF) produced 3.3 ng/ml IL-10 per 1
x 106 cells per 24 h in contrast to noninfected
tsF or tsF cells infected with a retrovirus coding for murine IFN-
where murine IL-10 production was undetectable. The sensitivity of the
ELISA was 15.0 pg/ml. Previously it has been shown that nontransduced
tsF cells do not produce detectable levels of endogenous IL-4, IL-10,
or IFN- as assessed by ELISA (11), or IFN-
or -
as assessed by anti-viral assay (20).
IL-10 CNS gene therapy in EAE
In three separate experiments, mice were injected i.c. on day
12 p.i. with either 2 x 106 IL-10.tsF
or nontransduced tsF cells. This was 1–2 days before the onset of the
first observable clinical signs but when the disease process is
ongoing, demonstrated by weight loss and infiltrate of inflammatory
cells to the CNS in EAE mice (12, 19). The inhibition of
EAE using IL-10.tsF was reproducible and the results from a typical
experiment are shown in Table I
. As a
negative control we have previously demonstrated that genetically
modified fibroblasts, coding for murine IFN- do not interfere with
the disease course of EAE (11). IL-10 delivered by
IL-10.tsF cells significantly reduced the mean clinical score of EAE
(1.8 ± 0.4; p < 0.005) compared with tsF-treated
mice (3.3 ± 0.3) although it had no effect on the mean onset of
disease (Table I). Most IL-10.tsF-treated mice did not progress above
grade 2 where they stabilized for 3–4 days before returning to mild
clinical disability of a slight loss of tail tone, grade 0.5, typical
in postacute remission animals. In contrast, control SCH and
tsF-treated mice progressed to maximum clinical severity, complete
hindlimb paralysis (grade 4), before returning to mild clinical
disease. Fig. 1 represents three
experiments pooled together and demonstrates the stabilized course of
EAE after CNS administration of IL-10.tsF compared with tsF-treated and
untreated SCH mice.
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The importance of the level of IL-10 delivered to the CNS was studied
by reducing the adenoviral titer so as to deliver approximately an
equivalent concentration of mouse IL-10 in vivo as from the IL-10.tsF.
In addition the concentration of recombinant murine IL-10 was also
reduced. However, adenoviral delivery of IL-10 from 5 x
106, 5 x 105, or
5 x 104 PFU AdRIL10 and direct
injection of 2 x 105, 5 x
103, 100, 10, and 1 ng/ml IL-10 injected i.c. day
12 p.i. all had no significant inhibitory effect on EAE
(Table I
).
IL-10 gene therapy reduces the perivascular lesion load in the CNS but increases CNS inflammatory cell infiltrate
In untreated EAE, the number of infiltrating cells closely correlates with the clinical score (19). Wax-embedded sections from the lumbar region of spinal cords from untreated SCH or tsF- or IL-10.tsF-treated groups were divided according to their EAE clinical score and the number of perivascular cuffs containing inflammatory cell infiltrate (perivascular lesions) assessed microscopically from three sections from each mouse per group. Each group contained six mice. Sections from untreated SCH mice at grade 4 had a mean lesion number of 14.3 ± 2.8 per section, where discrete perivascular cuffs were present in parenchyma of the spinal cord, with infiltrate both in the parenchyma and in the meninges. Spinal cord from tsF-treated mice at grade 4 had a mean lesion count of 11.5 ± 1.3 per section, and displayed similar pathology to the SCH group. Sections from IL-10.tsF-treated mice at grade 4 had a lower mean lesion number of 4.7 ± 1.3. However, despite a lower lesion count there appeared to be higher levels of infiltrate in the parenchyma surrounding the meninges and the meninges itself, which was not localized to perivascular tissue and did not form discrete lesions.
The mean lesion number from untreated SCH and tsF-treated mice at
clinical grade 2 were 6.8 ± 1.0 and 13.3 ± 2.9 per section
respectively. In contrast the IL-10.tsF-treated mice at grade 2 had a
significantly lower mean lesion number of 2.2 ± 0.5 per section
(p < 0.01). The untreated SCH and tsF-treated
spinal cords shared similar pathology with perivascular lesions in the
parenchyma and with inflammatory infiltrate widespread throughout the
parenchyma and meninges (Fig. 2A). Again the
IL-10.tsF-treated mice at grade 2 had an increased inflammatory
infiltrate in the meninges and surrounding parenchyma but with few
discrete perivascular lesions (Fig. 2B). In some instances
large numbers of infiltrating cells were seen in the meninges but not
infiltrating the spinal cord parenchyma. IL-10.tsF-treated mice, which
had stabilized disease for at least 3 days resulting only in loss of
tail tone (grade 1), had a mean lesion number of 0.6 ± 0.2. In
contrast, tsF-treated mice at grade 1, which typically had been
observed to continue the disease course to maximum disability (total
hind limb paralysis, grade 4) had a mean lesion number of 6.6 ±
2.6. However, as with the IL-10.tsF-treated mice at grade 2, the spinal
cord of IL-10.tsF-treated mice contained large numbers of cells in the
meninges but not invading the perivascular space or parenchyma.
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Immunofluorescent detection of the MHC class II Ag in cryostat
sections from the spinal cord from untreated SCH and tsF-treated mice
at grade 4 and grade 2 showed MHC class II+ cells
in perivascular lesions and notably within the surrounding parenchyma,
which corresponded to CD11b+ (i.e.,
macrophages/microglia) cells in the same areas (data not shown).
However, there was little or no positive MHC class II Ag staining in
the IL-10.tsF-treated group at grade 2 on resident cells within the
parenchyma although MHC class II+ cells could be
seen around the perivascular lesions and in infiltrate in the meninges
(Fig. 3).
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A previous immunocytochemical study in the ABH mouse during the
evolution and resolution of acute EAE showed a constant ratio of
CD4+ to CD8+ cells in the
spinal cord at 
8–9:1 (19). Likewise, in this study,
flow cytometry demonstrated an 8:1 ratio of
CD4+:CD8+ of CNS
inflammatory cells in tsF and untreated mice. In contrast this ratio
was reduced to 4:1 in IL-10.tsF disease-stabilized mice. This was
also supported by immunocytochemistry in frozen sections (Fig. 4). The numbers of
CD8+ T cells were low in sections from SCH and
tsF, yet there was a significant (p < 0.002)
increase in the number of CD8+ cells present in
the parenchyma and especially the meningeal infiltrate of spinal cords
taken from IL-10.tsF-treated mice stabilized at grade 2 compared with
all other groups. In addition, IL-10.tsF-treated mice with no clinical
disease observed also showed an increase in the number of
CD8+ T cells in the meninges (data not
shown).
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). Spinal cords from untreated SCH mice and tsF-treated mice at
clinical grade 2 had comparable numbers of CD4+
cells to each other but less than observed in the same groups at grade
4 (Fig. 4). In contrast, in IL-10.tsF-treated mice at grade 2 showing
inhibition of EAE there was a significant increase in the number of
CD4+ cells compared with the tsF-treated or
untreated SCH mice at grade 2 (p < 0.01) (Fig. 4). However, these mice had stabilized at grade 2 for 3 days compared
with tsF-treated and control SCH mice, which were sacrificed at first
signs of grade 2 clinical disease. Therefore, they were sacrificed at a
timepoint equivalent to that of grade 4 in tsF-treated and control SCH
mice, which is supported by a similar quantity of
CD4+ cells in the spinal cord of grade 4 mice
(Fig. 4). Interestingly, in IL-10.tsF-treated mice that did not exhibit
clinical disease at the time-point where control mice had maximum
paralysis, CD4+ T cells were present in the
meninges (data not shown).
Spinal cord sections analyzed at maximum clinical severity from
untreated SCH and tsF-treated mice had low levels of
B220+ cells (Fig. 4). IL-10.tsF-treated mice
stabilized at grade 2 exhibited a significantly greater number of
B220+ cells present in the meninges and
surrounding parenchyma compared with untreated SCH or tsF-treated mice
at grade 2 and 4 (p < 0.0005) (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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Injection of a wide dose range (2 x 105–1 ng/ml) of rIL-10 protein into the CNS before disease onset had no effect upon EAE disease course or severity, possibly due to lack of sustained effect, as it was not feasible to perform repeated i.c. injections, and the rapid clearance of the protein. Under the same conditions as this study we have demonstrated that i.c. injection of plasmid DNA-cationic liposome complex (CLC) induces limited expression of transgene over a short period of time and Il10-DNA-CLC has no effect upon EAE, probably due to low-level expression (24). In contrast to plasmid DNA-CLC it has been demonstrated that adenoviral gene delivery can produce a large quantity (high nanogram/microgram per milliliter) of transgene product over a 1- to 2-wk period, before its elimination by the immune response (16, 25). The high adenoviral production of IL-10 detected in vivo may induce endogenous feedback mechanisms to limit these unphysiological levels of IL-10 in the CNS and may explain the lack of efficacy in AdRIL10-treated animals. IL-10-expressing tsF have been shown to produce levels of IL-10 in the low nanogram per milliliter range in vivo and a previous study using fibroblasts retrovirally engineered to produce dTNFR demonstrated that these cells produced effective concentrations of transgene for at least up to 5 wk post implantation (11). This suggests that the level of IL-10 produced by the gene vector may be critical in determining whether Il10 gene therapy is successful or not. Therefore, the adenoviral titer was reduced to produce similar levels of IL-10 in the CNS in vivo as the IL-10.tsF. However, EAE was still not inhibited. Adenoviral delivery of viral IL-10 has been shown to be effective in collagen-induced arthritis, but in this circumstance was delivered systemically, where the environment may not be as sensitively regulated as the CNS and where the volume of body compartments will reduce the level of IL-10 reaching the target organ (26, 27). The duration of expression of a physiological concentration of IL-10 produced by the retroviral vector appears to be more beneficial than a sudden increase of high levels of IL-10 as seen with adenovirus and large bolus protein administration. Although adenovirus has low clinical value, the kinetic profile of "adenoviral-like" vectors may be more suitable for delivery of neutralizing agents such as Abs or fusion proteins, rather than cytokines.
The difference in efficacy of IL-10 delivered by immortalized
fibroblasts (tsF) in EAE compared with other vectors may also be due to
synergy between IL-10 and an unknown factor secreted by the tsF.
Although we have not detected endogenously produced IL-4, IL-10, or
IFN- or - in unstimulated, cultured nontransduced tsF or
IL-10.tsF, these cells produce TGF-, a cytokine that has been shown
to inhibit EAE (11, 24, 28). Neutralization of either
IL-10 or TGF- may determine whether there is synergy between these
cytokines in ameliorating EAE. However, neutralizing IL-10 from
IL-10.tsF with IL-10-specific mAb induces mortality in ABH mice when
injected i.c. (24). Similarly the use of Abs to TGF-
would neutralize both the TGF- produced by the tsF and endogenous
TGF- in the CNS. Therefore, we investigated whether secreted
products from the tsF cells could synergize with adenoviral-delivered
IL-10 and increase its efficacy in treating EAE. Adenoviral-delivered
genes exist epichromosomally and are lost during cell division, which
means that they are not appropriate vectors to make stable ex vivo cell
lines. To study the possible synergy, we investigated the
coadministration of nontransduced tsF cells and AdRIL10
injected i.c. day 12 p.i., yet this failed to affect the disease
course. Although this may indicate that synergy of factors such as
TGF- produced by tsF with IL-10 may not be the cause of the
increased efficacy of tsF-delivered IL-10, it is also possible that the
effect depends on the nature of how IL-10 affects the intracellular
function of the cell vector itself.
Other studies of IL-10 gene therapy in EAE have been inconclusive. EAE could be inhibited by memory T cells genetically modified with cDNA coding for IL-10 but not by MBP-specific T cell hybridoma cells retrovirally engineered to produce IL-10 (22, 23). The in vitro levels of IL-10 production from the hybridoma cells were 100 times greater than from the memory T cells (22, 23). However, both studies have a disadvantage in that numbers of CNS Ag-specific cells trafficking to the CNS have been shown to be low (1–4%) (29), and following in vivo transfer hybridoma cells proliferate and, therefore, the actual quantity of IL-10 delivered in vivo to the CNS cannot be estimated accurately. In addition, a recent study reports the inhibition of EAE using IL-10 secreting, non-CNS Ag-specific Th2 cells administered systemically (30). This suggests that the therapeutic action of neuroantigen-specific cells may not be confined to the CNS. In contrast, IL-10.tsF cells do not proliferate in vivo (as the temperature-sensitive SV40 large T Ag is down-regulated at mouse body temperature) and, therefore, a more reproducible dose of IL-10 can be administered directly to the CNS than with hybridoma cells. This is supported by the different histological profile seen in the CNS using IL-10.tsF and other studies administering IL-10 or IL-10-secreting vectors systemically (6, 8, 23, 30). The contradictory reports of IL-10 therapy in EAE may be due to variations in the models and routes of administration used and whether IL-10 exerts its effects in the peripheral compartment or the CNS, the target organ of EAE. However, it is also possible that such differences are reflective of the different cell types used and how the intracellular IL-10 may influence the other cytokines or cell surface molecules that the cell vectors express.
Long-term delivery of IL-10 to the CNS also allows the study of the
mechanism by which IL-10 may inhibit EAE at the target organ rather
than the effects of IL-10 on peripheral lymphoid organs seen by
systemic IL-10 administration. The histological profile in the CNS of
IL-10.tsF-treated animals exhibited a large number of inflammatory
cells accumulated within the meninges and underlying tissue. It was
evident that microglial activation had not been initiated, as
represented by the up-regulation of MHC class II Ags, in animals
injected with IL-10.tsF. This may reflect a direct action of cytokines
in inhibition of parenchymal microglia/macrophage function, or that the
release of pro-inflammatory T cell products such as IFN-, which is a
potent up-regulator of adhesion, costimulatory molecules and MHC class
II Ags was prevented thus limiting further expansion of the lesion.
Furthermore IL-10 may also regulate other molecules such as
metalloproteases, chemokines and adhesion molecules, which may be
required for extravasation across the vessel basement membranes and
into the parenchyma (31, 32, 33, 34, 35, 36, 37).
This study has demonstrated that IL-10.tsF significantly inhibited EAE when administered locally to the CNS. IL-10.tsF gene therapy consistently stabilized the majority of treated animals at clinical grade 2 of EAE (impaired righting reflex) for 3–4 days before the clinical score returned to baseline. This was not associated with inhibition of cellular recruitment but a change in the phenotype of inflammatory cells. There was a significant increase in frequency of CD8+ T cells and B cells in IL-10.tsF-treated mice. This supports previous work, which has shown that IL-10 can promote the growth of activated CD8+ cells (38, 39) and that increased pancreatic infiltration of CD4+, CD8+ T and B cells were observed in an IL-10 transgenic model in NOD mice (40, 41). In contrast to both the transgenic NOD study and the results we present here, transgenic mice constitutively producing human IL-10 (400–700 pg/ml in serum) were protected from the induction of EAE and failed to show histological evidence of CNS infiltration (42). Further study of the immune cells infiltrating the CNS as a result of IL-10 gene therapy may suggest the induction of "regulatory cells" in the inhibition of EAE.
This study has shown the benefit of local implantation of IL-10-producing immortalized fibroblasts to the CNS, to consistently and significantly reduce severity of EAE from a single injection. In addition this study has highlighted the importance of selecting the correct vector for the therapeutic gene to be administered. In the case of cytokine therapy little is known about the relative levels of cytokines in vivo and the feedback mechanisms, which control production and inhibition, especially in the CNS. Therefore, although in vitro experiments play an important role in determining potential cytokine function this study highlights the importance for in vivo experiments in animal models to determine the role of cytokines in their local environment. The increased inflammatory infiltrate observed in IL-10-treated spinal cord highlights a potential limitation associated with cytokine therapy in the CNS. Cytokines often have "pleiotropic" properties and chronic expression may result in many different responses. Although severity of disease was significantly reduced by IL-10 gene delivery from retrovirally engineered fibroblasts, chronic expression of "beneficial" cytokines may result in exacerbation of disease or other pathologies. Through the use of inducible promoters to regulate expression and timing of protein delivery, it may be possible to investigate this further.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Ludovic Croxford, Neuroinflammation Group, Department of Neurochemistry, Institute of Neurology, University College London, 1 Wakefield Street, London WC1N 1PJ U.K.
3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; SCH, spinal cord homogenate; CSF, cerebrospinal fluid; CLC, cationic liposome complex; AdRL, type 5 human adenovirus coding for LacZ from Escherichia coli; AdRIL10, type 5 human adenovirus coding for mouse IL-10; i.c., intracranially; p.i., postinoculation; tsF, temperature-sensitive fibroblast.
Received for publication February 8, 2000. Accepted for publication January 11, 2001.
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TopAbstractIntroductionMaterials and MethodsImmortalized fibroblast...Local administration and...Isolation of immune infiltrate...Histology of spinal cord...ImmunohistochemistryStatistical analysisResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or injected i.c. on day 12 p.i. with either 2
x 106 immortalized fibroblasts (tsF) (•) or
tsF-producing IL-10 (IL-10.tsF) (
). The data shows the inhibition of
EAE in mice treated with IL-10.tsF cells (mean clinical score =
2.2 ± 0.2) compared with tsF-treated (mean clinical score =
3.4 ± 0.2) or untreated mice (mean clinical score = 3.8
± 0.2). The results represent the mean clinical score ± SEM of
all animals within the group (n = 20–30).
). B, Inhibition of EAE by i.c.
injection of IL-10.tsF is associated with a decrease of OX-6 on
resident CNS cells but not on infiltrating cells present in the
meninges and perivascular lesions (