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*
Experimental Neuroimmunotherapy Unit, Department of Biotechnology, and
Department of Neurology, San Raffaele Scientific Institute, Milan, Italy;
Roche Milano Ricerche, Milan, Italy;
§
Department of Neurology, Casa Sollievo della Sofferenza Scientific Institute, San Giovanni Rotondo (FG), Italy;
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Howard Huges Medical Institute, Yale University School of Medicine, New Haven, CT 06510; and
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Vertex Pharmaceuticals, Inc., Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, IFN-
, and IL-6. A reduction of EAE incidence and severity
is observed in caspase-1-deficient mice, depending on the
immunogenicity and on the amount of the encephalitogenic myelin
oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient
mice, reduced EAE incidence correlates with defective development of
anti-MOG IFN--producing Th1 cells. Finally, pharmacological
blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord
homogenate or MOG35–55 peptide, by the caspase-1-inhibitor
Z-Val-Ala-DL-Asp-fluoromethylketone,
significantly reduces EAE incidence in a preventive but not in a
therapeutic protocol. These results indicate that caspase-1 plays an
important role in the early stage of the immune-mediated inflammatory
process leading to EAE, thus representing a possible therapeutic target
in the acute phase of relapsing remitting MS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and TNF-, although neither
IL-1 nor IL-6 nor TNF- are substrates for caspase-1
(5). Furthermore, caspase-1 proteolytically activates
IL-18, and caspase-1-/- mice have also reduced
serum levels of IL-18 and IFN- in response to LPS administration
(6). Caspase-1-/- mice are
resistant to LPS-induced endotoxic shock (7) and to the
induction of experimental pancreatitis (8). In vivo
pharmacological inhibition of caspase-1 protects mice from
TNF--induced liver failure (9) and collagen-induced
arthritis (10).
MS3 is an
immune-mediated demyelinating disease of the CNS of unknown etiology
(11). The pathological hallmark of the disease is the
presence within the CNS of inflammatory infiltrates containing few
autoreactive T cells and many pathogenic nonspecific mononuclear cells
(12). It is currently believed that Ag-specific T cells
provide the organ specificity of the pathogenic process and regulate
the recirculation within the CNS of activated mononuclear cells
releasing inflammatory myelinotoxic substances. These latter cells can
be activated in the periphery by polyclonal inflammatory stimuli, thus
determining disease recurrence (12, 13). Proinflammatory
cytokines participate either in Ag-specific T cell activation or in
peripheral activation of nonspecific mononuclear cells. TNF-,
IFN-, and IL-6 levels increase before disease relapses (13, 14). An increased number of disease relapses was observed in MS
patients treated with IFN- (15). Moreover, TNF-,
IFN-, and IL-1ß are present in demyelinating plaques
(16), and IL-1ß has been shown to be a mediator of the
inflammatory process sustaining EAE, the animal model for MS
(17).
We evaluated the role of caspase-1 in EAE. We found that caspase-1 mRNA
blood levels parallel those of proinflammatory cytokines, such as
IL-1ß, IL-6, TNF-, and IFN-, during EAE and peak at the time of
maximal EAE severity. A reduction of EAE incidence and severity was
observed in caspase-1-/- mice depending on the
immunogenicity and on the amount of the encephalitogenic MOG peptide
used. Finally, pharmacological blockade of caspase-1 reduced the
incidence of EAE, induced either with SCH or
MOG35–55 peptide, in a preventive but not
therapeutic protocol. These results indicate that caspase-1 plays an
important role in the immune-mediated pathogenic events leading to EAE
and might represent a suitable therapeutic target of the active phase
of the immune-mediated inflammatory demyelination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Biozzi AB/H mice, 4–6 wk old, were purchased from Harlan U.K. (Blackthorn, U.K.). Female C57BL/6 mice, 4–6 wk old were obtained by Charles River (Calco, Italy). Female (SV129 x C57BL/6)F1 mice, hereafter designated (SV129 x B6)F1, 4–6 wk old, were obtained by The Jackson Laboratory (Bar Harbor, ME). Caspase-1-/- mice had been obtained as previously described (5). Briefly, chimeric mice were obtained by injection of embryonic stem cells, in which the caspase-1 gene was disrupted and replaced with a neomycin resistance gene cassette, into C57BL/6 blastocysts. The chimeric males were then mated with CB57BL/6 mice. Homozygous mice with two copies of the disrupted caspase-1 gene were identified by Southern blot of genomic DNA, and the absence of caspase-1 mRNA in caspase-1-/- mice was confirmed by RT-PCR analysis. Homozygous mice were then interbred and used for the experiments.
All animals were housed in specific pathogen-free conditions and treated according to the guidelines of the Animal Ethical Committee of our Institute. Mice were immunized with IFA (Difco, Detroit, MI) supplemented with 4 mg/ml Mycobacterium tuberculosis (strain H37Ra; Difco) and MOG35–55 (Multiple Peptide Systems, San Diego, CA), MOG40–55 (Roche Milano Ricerche, Milan, Italy), or SCH from Biozzi AB/H mice. Two immunization schedules were used for peptides: a single injection of 200 µg or two injections of 300 µg peptide 7 days apart. For SCH immunization, 1 mg of Ag was given twice, at days 0 and 7. All injections were followed by i.p. administration of 500 ng pertussis toxin (Sigma, St. Louis, MI) the same day and 48 h later. Body weight and clinical score (0 = healthy, 1 = flaccid tail, 2 = ataxia and/or paresis of hind limbs, 3 = paralysis of hind limbs and/or paresis of forelimbs, 4 = tetraparalysis, 5 = moribund or death) were recorded daily.
Miniosmotic pumps
Miniosmotic pumps (Alzet 2001, Alza, Palo Alto, CA) were
implanted s.c. in the dorsal flank of mice. The mean fill volume of
pumps was 
220 µl, and the mean pumping rate was 1 µl/h,
delivering continuously for 10 days. Pumps were filled with 50 mg/ml
of the caspase-1 inhibitor
Z-Val-Ala-DL-Asp-fluoromethylketone
(Bachem, Bubendorf, Switzerland) obtaining a delivery of 1.2 mg/day.
Pumps were implanted 1 day before immunization (preventive protocol) or
1 week after (therapeutic protocol). Dosage of
Z-Val-Ala-DL-Asp-fluoromethylketone
has been deducted from previous reports showing that in vivo
administration of 25–50 mg/kg of specific caspase-1 inhibitors (i.e.,
Z-Val-Ala-DL-Asp-fluoromethylketone,
VE 13.045) completely inhibits caspase-1 enzymatic activity for several
days (18, 19).
Semiquantitative RT-PCR for cytokines
Blood samples were obtained from mice by tail bleeding every
week. RNA was recovered from these samples in guanidinium thiocyanate
by acid phenol extraction. A T-primed first strand kit was used for the
reverse transcription of total RNA into cDNA (Ready-to-go kit,
Pharmacia, Uppsala, Sweden). PCR amplification (30 cycles: 1 min
95°C, 1 min 55°C, 1 min 72°C) of cDNA sequences specific for
caspase-1 and cytokines was performed using 20 pmol of each primer, 200
mM concentrations of each dNTP, 25 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5
mM MgCl2, and 2.5 U Taq polymerase.
Amplified PCR products were hybridized with the specific
32P-labeled oligonucleotide probe (caspase-1 and
cytokines) or 32P-labeled probe obtained from a
plasmid containing the mouse GAPDH cDNA, followed by analysis on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA; Image Quant
Software, version 3.3). Values were normalized against the GAPDH gene.
For interanimal comparisons, the normalized intensities were further
corrected with the use of the normalized intensities of the bands
resulting from RT-PCR amplification of a cDNA derived from
mouse LPS-activated splenocytes (positive control). The following
primers and probes were used: caspase-1 (product: 343 bp), antisense
5'-GTGTTGAAGAGCAGAAAGCA-3', sense 5'-GAGATGGTGAAAGAGGTGAA-3',
probe 5'-TGAAAGACAAGCCCAAGG TG-3'; IL-1ß (product: 563 bp), antisense
5'-CAGGACAGGTATAGATTCTTTCCTTT-3', sense
5'-ATGGCAACTGTTCCTGAACTCAACT-3', probe
5'-AGCTTTCAGCTCATATGGGTCCGACAGCAC-3'; IL-6 (product: 634 bp),
antisense 5'-CACTAGGTTTGCCGAGTAGATCTC-3', sense
5'-ATGAAGTTCCTCTCTGCAAGAGACT-3', probe
5'-CTCCAGAAGACCAGAGGAAATTTCAATAG-3'; TNF- (product: 373 bp),
antisense 5'-GTATGAGATAGCAAATGCGCTGACGGTGTGGG-3', sense
5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3', probe
5'-GCCGTTGGCCAGGAGGGCGTTGGCGCGCTG-3'; IFN- (product: 450 bp),
antisense 5'-ACACTGCATCTTGGCTTTGC-3', sense
5'-CGACTCCTTTTCCGCTTCCT-3', probe 5'-TTCTTCAGCAACAGCAAGGC-3';
GAPDH (product: 710 bp), antisense 5'-CGCATCTTCTTGTGCAGTG-3'; sense
5'-GTTCAGCTCTGGGATGAC-3'. RT-PCR results were expressed as fold
induction of AU from basal levels.
Ag-specific proliferation assays
For T cell proliferation assays, draining lymph nodes were removed, and 4 x 105 lymph node cells per well were cultured in 96-well culture plates (Costar, Cambridge, MA) in synthetic HL-1 medium (Ventrex Laboratories, Portland, ME) supplemented with 2 mM L-glutamine and 50 µg/ml gentamicin (Sigma, St. Louis, MO) and serial concentrations (1, 3, 10 µM) of MOG40–55 peptide. Cultures were incubated for 3 days in 5% CO2 in air and pulsed 8 h before harvesting with 1 µCi [3H]TdR (40 Ci/nmol, Pharmacia Biotech, Amersham, Cologno Monzese, Italy). Incorporation of [3H]TdR was measured by liquid scintillation spectrometry.
Intracytoplasmatic staining for cytokine production
Lymph node cells (6 x 105
cells/well) were cultured in 96-well culture plates in synthetic HL-1
medium with 10 µM MOG40–55. After 72 h of
culture cells were harvested, washed, and recultured for additional
72 h in RPMI 1640 supplemented with 2 mM L-glutamine,
50 µg/ml gentamicin, 50 µM 2-ME (Fluka Chemical, Ronkonkoma, NY),
and 10% FCS (Sigma). After culture, living cells separated on a Ficoll
gradient were restimulated with PMA (1 µg/ml) and ionomycin (50
ng/ml) for 4 h at 37°C, with 10 µg/ml brefeldin A (Novartis,
Basel, Switzerland) added for the last 2 h to prevent egress of
newly synthesized proteins from the endoplasmic reticulum. After
fixation with 4% paraformaldehyde for 20 min at room temperature,
cells were stained for IFN- and IL-4 using the method of Openshaw et
al. (20) and Galbiati et al. (21). Cells were
washed, preincubated for 10 min with PBS/FCS/saponin, and then
incubated with FITC rat anti-mouse IFN- (XMG1.2, PharMingen, San
Diego, CA) and PE rat anti-mouse IL-4 (11B11, PharMingen) or with
isotype controls FITC- and PE-labeled rat IgG1, 
(R3-34,
PharMingen). After 30 min, cells were washed twice with PBS/FCS/saponin
and then with PBS containing 5% of FCS without saponin to allow
membrane closure. Cell membranes were then stained with
Cy-Chrome-labeled anti-CD4 (L3T4, PharMingen) for 15 min at room
temperature. Analysis was performed with a FACScan flow cytometer
(Becton Dickinson, Mountain View, CA) equipped with CellQuest software,
and 50,000 events were acquired.
ELISA for IFN- secretion
IFN- was quantified by two-sites sandwich ELISA using
polyvinyl microtiter plates (Falcon 3012) coated with AN-18.17.24 mAb
in carbonate buffer as previously described (21). Samples
(50 µl/well) diluted in test solution (PBS containing 5% FCS and 1
g/l phenol) were incubated together with 50 µl peroxidase-conjugated
XMG1.2 mAb. After overnight incubation at room temperature, bound
peroxidase was detected by 3,3',5,5'-tetramethylbenzidine (Fluka
Chemical), and adsorbance was read at 450 nm with an automated
microplate ELISA reader (MR5000, Dynatech Laboratories, Chantilly, VA).
IFN- was quantified from two to three titration points using
standard curves generated by purified recombinant mouse IFN-, and
results were expressed as cytokine concentration in ng/ml.
Detection limit was 15 pg/ml.
Neuropathological features in EAE mice
At the time of sacrifice, mice were transcardially perfused with 4% paraformaldehyde. Brains and spinal cords were removed and postfixed in the same fixative for 2–4 h, washed in PBS, and then embedded in paraffin. Tissue sections were cut at 4 µm on a microtome and stained for histological examination. Hematoxylin and eosin staining was used to reveal perivascular inflammatory infiltrates, Luxol Fast Blue staining was used to reveal demyelinated areas, and Bielshowsky staining was used to detect axonal loss. Macrophages were stained using peroxidase-labeled BS-I isolectin B4 (Sigma), whereas T cells using a rat anti-CD3 Ab (pan-T cell marker; Serotec Ltd, Oxoford, U.K.) revealed using a biotin-labeled secondary anti-rat Ab (Amersham). Neuropathological findings were quantified on an average of 10 complete cross-sections of spinal cord per mouse. The number of perivascular inflammatory infiltrates was calculated and expressed as the numbers of inflammatory infiltrates per mm2, whereas demyelinated areas and axonal loss were expressed as the percentage of damaged area per mm2 (22). T cells and macrophages were counted and expressed as the number of cells per mm2.
Statistical analysis
Data are expressed as mean ± SE. Student’s t
test for unpaired data was used to compare cytokine and caspase-1 mRNA
levels and the cytokine protein levels. EAE cumulative score was
calculated by summing up each individual score registered in any group
of mice studied during the follow-up period levels. Comparison between
cumulative scores was performed using Student’s t test. A
2 test was used to compare EAE incidence in
the different groups of mice. p < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice immunized with MOG40–55 were
bled once a week for 5 weeks after immunization. RT-PCR was performed
on blood samples to determine caspase-1, IL-1ß, IL-6, TNF-, and
IFN- mRNA levels. Caspase-1 mRNA levels increased almost 8-fold
(Fig. 1
A) during EAE from
basal levels recorded at day 7 p.i. This increase paralleled
disease severity, peaking 4 wk p.i. (day 27 p.i., 52.9 ±
16.8 AU; p = 0.01 vs day 7 p.i.) (Fig. 1A) when EAE clinical score reached its maximum (Fig. 1C). Caspase-1 mRNA level increase paralleled that of the
caspase-1 substrate IL-1ß, which showed a 2-fold increase (day
27 p.i., 3.3 ± 0.9 AU; p = 0.03 vs day
7 p.i.) (Fig. 1B); IL-6 mRNA increased almost 8-fold
(day 27 p.i., 15.4 ± 7.6 AU; p = 0.04) (Fig. 1B); TNF- mRNA increased almost 3-fold (day 27 p.i.,
12.2 ± 5.4; p = n.s. vs day 7 p.i.) (Fig. 1B), and IFN- mRNA 4-fold (day 27 p.i., 1.6 ±
0.5 AU; p = 0.02 vs day 7 p.i.) (Fig. 1B). These data indicate that caspase-1 is up-regulated at
the transcriptional level during thecourse of EAE and that this
up-regulation parallels that of proinflammatory cytokines such as
IL-1ß, IFN-, TNF-, and IL-6.
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To further explore the role of caspase-1 in EAE, we studied the
susceptibility of caspase-1-/- mice to MOG
peptide-induced EAE. We first analyzed EAE susceptibility of
(SV129 x B6)F1 and C57BL/6 mice, which have
a genetic background comparable with that of the
caspase-1-/- mice using 200 µg of
MOG35–55. The two groups of mice showed similar
disease courses (Fig. 2A,
Table I) confirming results previously
reported (23).
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B, Table I).
Caspase-1-/- mice developed EAE when immunized
with the low dose (200 µg) of MOG35–55 (Fig. 2A, Table I) or the high dose (600 µg) of
MOG40–55 (Fig. 2B, Table I); the
incidence, however, was lower and the onset delayed compared with
(SV129 x B6)F1 or C57BL/6 mice. Therefore,
caspase-1-/- mice are less susceptible to
MOG-induced EAE compared with C57BL/6 or (SV129 x
B6)F1 mice. However,
caspase-1-/- mice can develop EAE when the
immunogenicity of the encephalitogenic peptide is increased. Inhibition of the Th1 response in caspase-1-deficient mice
To elucidate the possible causes of reduced susceptibility of
caspase-1-/- mice to EAE, we analyzed mice
immunized with 200 µg of MOG40–55 peptide
in which the most striking difference between
caspase-1-/- and C57BL/6 was observed (Fig. 2B, Table I). Five caspase-1-/-
mice and 5 C57BL/6 mice were sacrificed 10 days after immunization with
MOG40–55, and cells from the draining lymph
nodes were obtained. Proliferation assays to
MOG40–55 showed no impairment in the
responsiveness of T cells from caspase-1-/-
compared with C57BL/6 mice (Fig. 3A). We detected, however,
decreased levels of IFN- secreted in the supernatant of
MOG40–55 restimulated cells (Fig. 3B) from
caspase-1-/- compared with C57BL/6 mice
(p = 0.01). Intracytoplasmic staining for
IFN- and IL-4 of MOG40–55-restimulated cells
showed reduced IFN--producing cells and no induction of
IL-4-producing cells (Fig. 3, C–E). These results suggest
that cells from caspase-1-/- mice efficiently
present the encephalitogenic peptide to T cells but are defective in
Th1 development.
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Pharmacological blockade of EAE with caspase-1 inhibitors was
tested in Biozzi AB/H mice immunized with SCH or
MOG35–55. In Biozzi mice, SCH induces a very
aggressive relapsing-remitting EAE (25), whereas
MOG35–55 induces a more chronic progressive disease. We
administered vehicle or
Z-Val-Ala-DL-Asp-fluoromethylketone,
a highly specific, cell-permeable, and irreversible inhibitor of
caspase-1-like proteases (26) to Biozzi mice before
immunization with SCH or MOG35–55. Because this
caspase-1 inhibitor is a peptide with a very short half-life in vivo,
we implanted s.c. mini-osmotic pumps able to continuously release the
inhibitor for 10 days. Continuous administration of the caspase-1
inhibitor induced a clearcut suppression of either SCH-induced or
MOG35–55-induced EAE compared with
vehicle-treated mice (Table II). The
cumulative EAE score, representing the disease burden, was
significantly lower in caspase-1 inhibitor-treated mice than in
vehicle-treated mice in both immunization protocols (Table II). However, the mean day of onset and the mean clinical score of sick
animals were comparable between groups (Table II). In the SCH-induced
group, two animals for each treatment were sacrificed for
neuropathology examination. Animals treated with the inhibitor had less
infiltrates per mm2 than did vehicle-treated
controls (0.4 and 1.6 vs 6.6 and 3.1, respectively) (Fig. 4, A and B), less
demyelination (0.6% and 0.8% vs 9.8% and 2.2%, respectively) (Fig. 4, C and D), reduced axonal loss (0.4% and 0.6%
vs 8.3% and 1.5%, respectively) (Fig. 4, E and
F), less infiltrating CD3+ cells/mm2
(39.9 and 90.5 vs 291 and 138.2, respectively), and reduced
numbers of infiltrating macrophages per mm2 (21.8
and 52.5 vs 211.9 and 84.9, respectively).
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). Only 1 of the 10 mice
treated with the inhibitor from 7 days p.i. did not develop a
full-blown EAE. These results suggest that caspase-1 is crucial in the
induction phase of EAE, when peripheral inflammation leads to the
recruitment of effector cells into the CNS. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, TNF-, IL-18, and IFN-
(5, 6, 7). We found that caspase-1 transcription is
up-regulated during mouse EAE, reaching a peak at maximal disease
severity. This induction is higher compared with other proinflammatory
cytokines previously shown to be up-regulated during EAE
(27) and is similar to that found in the Lewis rat EAE
(30). These data suggest that proteolytic activation of
proinflammatory cytokines is a crucial step in the immune-mediated
process leading to EAE.
Caspase-1-/- mice were susceptible to MOG
peptide-induced EAE. Disease susceptibility was associated with the
number of immunizations and the dose and the MHC-binding affinity of
the encephalitogenic MOG-peptide. In our hands,
MOG40–55 showed a 3-fold weaker binding
(IC50 = 360 pM) compared with
MOG35–55 (IC50 = 1 nM) to
the I-Ag7 molecule (data not shown). However, the
disease induced in caspase-1-/- mice was always
less severe then in C57BL/6 or (SV129 x
B6)F1 mice and did not develop at all when a low
dose of the weaker MHC binder MOG peptide was used. The reduced EAE in
caspase-1-deficient mice was not caused by inefficient Ag presentation
to T cells, as shown by the Ag-specific proliferation assays, but to an
impairment in Th1 cell development as indicated by the lower percentage
of IFN--producing cells from caspase-1-/- vs
C57BL/6 mice. The most likely explanation of this finding possibly
resides in the cleavage activity of caspase-1 on the precursor of IL-18
into its mature form (6). Although IL-18 in itself does
not induce Th1 cell development (31, 32), which is mostly
driven by IL-12 (33), Th1 cell development independent
from IL-12 could be induced by the cooperative action of IL-18 and
other factors (34). These factors may include IFN-
itself (35) and IL-1ß (33). Thus, the
simultaneous caspase-1-dependent defect in IL-18 and IL-1ß
production could explain the impaired Th1 development and the reduced
EAE in caspase-1-/- mice. A more aggressive
immunization protocol, however, is able to overcome this limiting step
in EAE induction, likely because other proteases may replace caspase-1
in the cleavage of pro-IL-1ß and pro-IL-18.
Results obtained with caspase-1 inhibitor administration are consistent with the lower susceptibility to EAE of caspase-1-/- mice. The preventive administration of the caspase-1 inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone dramatically reduced both relapsing-remitting or chronic-progressive EAE in two different mouse strains. However, the small proportion (35%) of mice not protected from EAE by caspase-1 inhibitor treatment developed a clinical course of EAE indistinguishable from that of controls. As in inflammatory fluids other proteases (i.e., trypsin, chymotrypsin, elastase, granzyme A) (36) can replace caspase-1 enzymatic activity, an immunization protocol able to induce a potent local inflammation may bypass its requirement. Nevertheless, recent data indicate that Z-Val-Ala-DL-Asp-fluoromethylketone is active also on other cysteine proteases involved in inflammatory reactions but not in the apoptotic cascade, such as caspases-4 and -5 (37).
Administration of Z-Val-Ala-DL-Asp-fluoromethylketone during the effector phase of SCH-induced EAE (i.e., therapeutic protocol) had no effect on the disease course and severity. These data are suggestive for a crucial role played by caspase-1 in the peripheral activation of proinflammatory cytokines during the induction phase of EAE. The lack of efficacy of caspase-1 inhibitors once the inflammatory cascade has already determined CNS infiltration and damage might be also explained by the impaired blood-brain barrier crossing of caspase-1 inhibitors (26).
In conclusion, our results indicate that caspase-1 plays a crucial role in the development of the immune-mediated inflammatory process leading to CNS demyelination. EAE development is impaired in caspase-1-/- mice, although the requirement for caspase-1 in the inflammatory phase of EAE can be bypassed. The role of caspase-1 in autoimmune demyelination is further corroborated by the significant reduction of EAE incidence in mice preventively treated with the caspase-1-inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone. However, the inefficacy of caspase-1 inhibitors in blocking ongoing EAE suggests that this enzyme is crucial in the early phase of the inflammatory process leading to immune-mediated demyelination. Thus, caspase-1 could represent both a marker of inflammation sustaining immune-mediated demyelination and a possible therapeutic target of the acute phase of relapsing-remitting MS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gianvito Martino, Department of Biotechnology-San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; SCH, spinal cord homogenate; AU, arbitrary units; p.i., postimmunization.
Received for publication April 12, 1999. Accepted for publication June 10, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Furlan, E. Brambilla, F. Ruffini, P. L. Poliani, A. Bergami, P. C. Marconi, D. M. Franciotta, G. Penna, G. Comi, L. Adorini, et al. Intrathecal Delivery of IFN-{gamma} Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes J. Immunol., August 1, 2001; 167(3): 1821 - 1829. [Abstract] [Full Text] [PDF]
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), IL-6 (
), IFN-
), and TNF-
) from the same mice. mRNA levels were measured
by a semiquantitative RT-PCR on blood samples obtained from the tail
vein of mice once a week. mRNA levels have been normalized against
GAPDH mRNA levels and expressed as fold induction (AU increase over
basal level recorded 7 days p.i.). Blood samples were obtained every
week after immunization as indicated on x-axis.
C, Clinical course of EAE in blood donors
(n = 12 mice). Each circle represents the mean EAE
score registered. Bars represent the SE.
= 200
µg MOG40–55; • = 600 µg MOG40–55).
Immunization of caspase-1-/- mice with the low amount
(200 µg) of the shorter peptide (MOG40–55) does not lead
to EAE development. Irrespective of the dose of MOG40–55
used, C57BL/6 mice always show a more severe EAE than do
caspase-1-/- mice.
cpm). B,
IFN-