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Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes1
*
Neuroimmunology Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy;
Department of Clinical and Experimental Medicine, Section of Microbiology, University of Ferrara, Ferrara, Italy;
Laboratory of Neuroimmunology, Neurological Institute "C. Mondino," University of Pavia, Pavia, Italy;
Roche Milano Ricerche, Milan, Italy; and
¶ Department of Neurology, San Raffaele Scientific Institute, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gene leads to persistent (up to 4 wk) CNS production of IFN-
and inhibits the course of a chronic-progressive form of experimental
autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin
oligodendrocyte glycoprotein (MOG)35–55. Mice treated with
the IFN--containing vector before EAE onset showed an earlier onset
but a milder course of the disease compared with control mice treated
with the empty vector. In addition, 83% of IFN--treated mice
completely recovered within 25 days post immunization, whereas control
mice did not recover up to 60 days post immunization. Mice treated with
the IFN--containing vector within 1 wk after EAE onset partially
recovered from the disease within 25 days after vector injection,
whereas control mice worsened. Recovery from EAE in mice treated with
IFN- was associated with a significant increase of CNS-infiltrating
lymphocytes undergoing apoptosis. During the recovery phase, the mRNA
level of TNFR1 was also significantly increased in CNS-infiltrating
cells from IFN--treated mice compared with controls. Our results
further challenge the exclusive detrimental role of IFN- in the CNS
during EAE/multiple sclerosis, and indicate that CNS-confined
inflammation may induce protective immunological countermechanisms
leading to a faster clearance of encephalitogenic T cells by apoptosis,
thus restoring the immune privilege of the CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and soluble TNFR1,
two molecules with anti-inflammatory properties, failed to achieve
therapeutically significant results in MS patients (4, 5).
In particular, TNFR1 therapy induced in some of the treated patients a
paradoxical proinflammatory effect, as indicated by the increased
number of inflammatory CNS lesions detected by gadolinium-enhanced
magnetic resonance imaging (MRI) (5). IFN-, the current
elective therapy for MS, reduces dramatically the number of
inflammatory CNS lesions but fails to achieve a satisfactory control of
the disease evolution (6). Similarly, s.c. administration
in MS patients of glatiramer acetate, a random copolymer able to induce
a specific T cell response with production of anti-inflammatory
cytokines, does not consistently affect the disease course
(7). The use of cladribine (8), a nucleoside
analog with anti-neoplastic activity in vitro and in vivo and
potently toxic to monocytes, and of Campath-1, a depleting anti-CD4
Ab (9), produced and sustained a significant reduction in
the number and volume of inflammatory brain lesions detected by
gadolinium-enhanced MRI in MS patients but did not affect the clinical
course of the disease.
The mixed results of anti-inflammatory therapies in MS and the
recent evidence showing that the presence of brain inflammatory lesions
is not a predictor of disability development in MS patients
(10), questions the exclusive detrimental role of
inflammation in this disease. Some experimental evidence also supports
this apparent paradox. C57BL/6J mice immunized with spinal cord
homogenate increased the morbidity rate of experimental autoimmune
encephalomyelitis (EAE) from 20 to 80% when treated with a
neutralizing mAb against IFN- (11). CNS-infiltrating T
cells from MS brains, as well as encephalitogenic T cells from EAE
mice, can secrete myelin-protective factors such as brain-derived
neurotrophic factor (12). CD4+ T
cells, both Th1 and Th2, can induce microglia to secrete
IL-12-inhibiting agents such as PGE2, thus
self-limiting the inflammatory process (13). In addition,
myelin basic protein-specific encephalitogenic T cells from EAE mice
protect from secondary demyelination possibly by fostering the
clearance of toxic substances released in situ following the injury
(14).
Here, we further challenge the concept of the exclusive detrimental
role of CNS inflammation in autoimmune demyelination by showing that
CNS-specific production of IFN-, a prototypical proinflammatory
cytokine, before or after EAE onset, can protect mice from disease
progression by inducing a fast clearance of encephalitogenic T cells
infiltrating the CNS parenchyma via an apoptotic pathway associated
with up-regulation of the TNFR1 death receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 female mice, 6–8 wk of age, were obtained from Charles River (Calco, Italy). Mice were housed in specific pathogen-free conditions, allowing free access to food and water. All procedures involving animals were performed according to the guidelines of the Animal Ethical Committee of our Institute (Institutional Animal Care and Use Committee).
HSV-1-derived vector preparation and injection
We used two different viral constructs (provided by J. Glorioso,
University of Pittsburgh, Pittsburgh, PA), which have been
previously described (15, 16). Briefly, the
IFN- gene-containing vector (d120:IFN-:LacZ
vector) accommodates the Escherichia coli LacZ
gene driven by the human CMV immediate early promoter and the mouse
IFN- gene driven by the infected cell protein 4 promoter.
The control vector (d120:LacZ vector) was identical with the
d120:IFN-:LacZ vector but contained only the
LacZ gene. Expression cassettes containing the heterologous
genes (i.e., LacZ, IFN-) were inserted in both vectors
into the thymidine kinase locus on a infected cell protein 4-defective
HSV-1 virus (d120) that was propagated on a complementing cell line
(E5). HSV-1 stocks were diluted with complete DMEM (supplemented with
10% FCS) to obtain 1 x 109 PFU/ml. Ten
microliters of the vectors (107 PFU) were
injected within the cisterna magna (intracisternally, i.c.) of
each mouse by using a 27-gauge stainless needle curved 40° as
previously described (16, 17).
EAE induction and treatment
EAE was induced by s.c. immunization in the flanks with a total
of 200 µg of myelin oligodendrocyte glycoprotein (MOG) aa35–55
(Multiple Peptide Systems, San Diego, CA) per mouse in IFA (Difco,
Detroit, MI) supplemented with 4 mg/ml of Mycobacterium
tuberculosis (strain H37Ra; Difco). Mice received 500 ng of
pertussis toxin (Sigma, St. Louis, MO) i.v. at the time of the
immunization and 48 h later. Weight and clinical score (0 =
healthy, 1 = flaccid tail, 2 = ataxia and/or paresis of
hindlimbs, 3 = paralysis of hindlimbs and/or paresis of forelimbs,
4 = tetraparalysis, 5 = moribund or death) were recorded
daily. Mice were i.c. injected with the IFN--containing vector or
with the control vector either before EAE onset (preventive protocol:
two injections at the day of immunization and 7 days later) or after
EAE onset (therapeutic protocol: one injection within 1 wk from disease
onset). Four C57BL/6 mice were immunized but remained untreated for the
whole follow-up period thus serving as controls.
Recovery of infiltrating cells from the CNS of EAE mice
Brain and/or spinal cord from EAE mice were homogenized with a potter in 2 ml of saline. Disrupted tissue has been brought to a volume of 10 ml and pelleted by centrifugation at 400 x g for 10 min. One milliliter 100% Percoll (Percoll; Pharmacia, Uppsala, Sweden, diluted 9/1 v/v with 10x PBS), 2 ml 80% Percoll, 3 ml 40% Percoll, and 2 ml 30% Percoll in PBS were stratified on the pellet. After centrifugation at 400 x g for 10 min, mononuclear cells were recovered from the interface between the 40 and 80% Percoll layers, washed several times, resuspended, and counted.
Blood brain barrier (BBB) permeability assessment
BBB permeability was assessed by measuring the total protein content in paired cerebrospinal fluid (CSF) and serum samples by a bicinchoninic acid protein assay commercial kit (Pierce, Rockford, IL). Serum samples were obtained from the tail vein and CSF was obtained from the cisterna magna using a 25-gauge needle. Data were expressed as ratio between CSF and serum protein content. Blood-contaminated CSF samples were discarded.
Semiquantitative RT-PCR for cytokine measurements
Total RNA was extracted from the CNS (brain and spinal cord)
from mice by the guanidinium thiocyanate method and then
retrotranscribed into cDNA by a random hexamer-primed commercially
available kit (Ready-to-go; Pharmacia). Total RNA was then tested for
cytokine and TNFR1 transcript level by semiquantitative RT-PCR. cDNA
synthesis, PCR amplification, and detection of amplified products were
performed as previously described (17). The following
primers and probes were used: IL-1 (product: 563 bp), antisense
5'-CAG GAC AGG TAT AGA TTC TTT CCT TT-3', sense 5'-ATG GCA ACT GTT CCT
GAA CTC AAC T-3', probe 5'-AGC TTT CAG CTC ATA TGG GTC CGA CAG CAC-3';
IL-4 (product 181 bp), antisense 5'-GAC TCA TTC ATG GTG CAG CTT ATC
G-3', sense 5'-CGA AGA ACA CCA CAG AGA GTG AGC T-3'; TNF-
(product:
373 bp), antisense 5'-GTA TGA GAT AGC AAA TGC GCT GAC GGT GTG GG-3',
sense 5'-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3', probe 5'-GCC
GTT CCG CAG GAG GGC GTT GGC GCG CTG-3'; TNFR1 (product: 297 bp),
antisense 5'-CCA TCC ACC ACA GCA TAC AG-3', sense 5'-GCC CCC TCC CCA
GCC TTC AG-3', probe 5'-CCC CAG CCT TCA GCC CCA CCT CCG G-3'; IFN-
(product: 450 bp), antisense 5'-ACA CTG CAT CTT GGC TTT GC-3', sense
5'-CGA CTC CTT TTC CGC TTC CT-3', probe 5'-TTC TTC AGC AAC AGC AAG
GC-3'; IL-6 (product: 634 bp), antisense 5'-CAC TAG GTT TGC
CGA GTA GAT CTC-3', sense 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3',
probe 5'-CTC CAG AAG ACC AGA GGA AAT TTT CAA TAG-3'; and GAPDH
(product: 710 bp), antisense 5'-CGC ATC TTC TTG TGC AGT G-3'; sense
5'-GTT CAG CTC TGG GAT GAC-3'. Values were normalized against the
GAPDH gene. RT-PCR results were expressed as arbitrary
units (AU).
T cell proliferation assay
Draining lymph node cells (LNC) were removed, and 4 x 105 cells/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) with different concentrations (1, 3, and 10 µM) of MOG35–55 or with 5 µg/ml of Con A. Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air and pulsed 8 h before harvesting with 1 µCi [3H]TdR (40 Ci/nmol; The Radiochemical Center, Amersham, Buckinghamshire, U.K.). Incorporation of [3H]TdR was measured by liquid scintillation spectrometry.
IFN- protein level measurement
IFN- was quantified by two-site sandwich ELISA using
polyvinyl microtiter plates (Falcon 3012) coated with AN-18.17.24 mAb
in carbonate buffer as previously described (16).
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, Ronkonkoma, NY), 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 nanograms per milliliter. Detection limit was
15 pg/ml.
Neuropathological analysis in EAE mice
At time of sacrifice mice were transcardially perfused with 4%
paraformaldehyde. Brains and spinal cords were removed and embedded in
paraffin. Tissue sections were cut at 10 µm and stained with
hematoxylin and eosin, Luxol fast Blue, and Bielshowsky stain to detect
inflammatory infiltrates, demyelination, and axonal loss, respectively.
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 number of
inflammatory infiltrates per square millimeter; demyelinated areas and
axonal loss were expressed as the percentage of damaged area per square
millimeter as previously described (16, 17). Vector
distribution within the CNS was traced by detecting HSV-1 with a rabbit
anti-HSV-1 polyclonal Ab (Accurate Chemical and Scientific,
Westbury, NY) followed by a biotinylated donkey anti-rabbit
Ig Ab (Amersham) whose signal was amplified by an avidin-biotin complex
(Vector Laboratories, Burlingame, CA). IFN- was revealed with a rat
anti-mouse IFN- Ab (PharMingen, San Diego, CA) followed by a
secondary biotinylated sheep anti-rat Ig Ab (Amersham) amplified
using an avidin-biotin complex (Vector Laboratories). T cells were
stained using a rat anti-CD3 (pan-T cell marker) (Serotec, Oxford,
U.K.), and TNFR1 with a rabbit anti-mouse TNFR1 polyclonal Ab
(provided by A. Corti, San Raffaele Scientific Institute, Milan,
Italy). Both Abs were revealed with a secondary biotinylated Ig Ab
(Amersham) and amplified using an avidin-biotin complex (Vector
Laboratories). Macrophages were detected using biotinylated BS-I
isolectin B4 (Sigma), and MHC class II molecules with a mouse
biotinylated anti-I-Ab mAb (PharMingen). Both
reactions were amplified using an avidin-biotin complex (Vector
Laboratories). The number of T cells and macrophages lining within the
subarachnoid space or infiltrating the CNS parenchyma was calculated
and expressed as the number of cells per square millimeter.
-galactosidase (-Gal) activity was detected by exposing tissue
sections to 1 mM
5-bromo-4-chloro-3-indolyl--D-galactoside for 3 h.
Detection of apoptotic cells
For detection of apoptotic cells, lympho-mononuclear cells isolated from the CNS were stained with annexin V FITC (PharMingen) and propidium iodide (PI) (50 µg/ml; Sigma). Cells were analyzed with a FACScan flow cytometer using CellQuest software (BD Biosciences, Oxfordshire, U.K.), as described (18). Apoptotic cells were detected on tissue sections using a commercially available immunohistochemical detection kit for TUNEL analysis (Roche, Mannheim, Germany). Double staining was also performed using a rat anti-CD3 (Serotec) revealed with a biotin-labeled secondary anti-rat Ab (Amersham).
Statistical analysis
EAE scores, RT-PCR, and neuropathological results were compared
using the Mann-Whitney nonparametric test and Student’s t
test for unpaired data. Frequencies between recovered vs nonrecovered
mice were compared using the 
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven days after i.c. injection into naive C57BL/6 mice, the
IFN--containing vector (d120:IFN-:LacZ) or the control
vector containing the LacZ gene only (d120:LacZ)
were found distributed in all ventricular (Fig. 1
, A–C) and subarachnoid
(Fig. 1, D–F) spaces, either rostrally or
caudally to the site of injection. HSV-1-derived vectors infected only
cells facing the CSF space, choroidal, ependymal (Fig. 1A),
and leptomeningeal cells (Fig. 1D), and not cells located
within the CNS parenchyma such as glial cells and neurons (Fig. 1).
Interestingly, vectors can also reach the CNS parenchyma from the
subarachnoid space along penetrating blood vessels, via the
Virchow-Robin spaces (Fig. 1, D and E).
d120:IFN-:LacZ vector-infected cells produced both
-Gal (Fig. 1, B and E) and IFN- (Fig. 1, C and F). IFN- production did not induce any
detectable pathological effect in the CNS or a recruitment of
blood-derived mononuclear cells (Fig. 1).
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CNS delivery of the IFN- gene induces in situ
production of IFN- and class II MHC expression but not BBB damage in
naive mice
C57BL/6 naive mice were i.c. injected twice (days 0 and 7) with
the d120:IFN-:LacZ and d120:LacZ
vectors and sacrificed 1 and 3 wk after the second injection. Five
untreated naive mice served as controls.
IFN--injected mice showed a significant (p
< 0.05) up-regulation in the brain and spinal cord of IFN- mRNA
compared with d120:LacZ-injected mice and untreated control
mice 1 wk after the second d120:IFN-:LacZ vector
injection (Fig. 2, A and
B). Three weeks after the second
d120:IFN-:LacZ vector injection, IFN- mRNA level was
higher compared with controls but was decreasing toward the baseline
(Fig. 2, C and D). Compared with untreated mice,
IL-1, IL-6, and TNF- mRNA levels were slightly but not
significantly higher in the brain and in the spinal cord of either
IFN--injected mice or d120:LacZ-injected control mice
(Fig. 2B). IL-4 mRNA was never found, either in untreated or
in treated mice (data not shown). Intracisternal delivery of the
IFN- gene-containing vector (Fig. 2F) but not
of the d120:LacZ control vector (Fig. 2E) induced
up-regulation of MHC class II molecules on leptomeningeal cells within
the subarachnoid space and on neuronal cells within the CNS
parenchyma.
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(range 43–243 pg/ml) were occasionally found in
serum samples from mice injected with d120:IFN-:LacZ (2
of 9) or with the d120:LacZ control vector (2 of 7). CSF and
serum protein content was measured in mice treated with the
d120:IFN-:LacZ or the d120:LacZ control vector
and in untreated mice to assess the BBB permeability.
Blood-contaminated CSF samples were discarded. No statistically
significant difference was found in the CSF and serum protein content
measured 1–3 wk after the second vector injection in
d120:IFN-:LacZ-treated mice (CSF: 178 ± 67 mg/dl,
range 44–365; serum: 3278 ± 292 mg/dl, range 1140–4890), in
d120:LacZ-treated mice (CSF: 95 ± 9 mg/dl, range
64–136; serum: 2499 ± 357 mg/dl, range 610-4770), and in naive
untreated mice (CSF: 273 ± 221 mg/dl, range 26–715; serum:
4496 ± 604 mg/dl, range: 2240–5720).
These results indicate that the i.c. injection of IFN--containing
vector induces a significant production of IFN- either in the brain
or in the spinal cord lasting at least 3 wk after vector injection.
Vector injection is initially accompanied by a slight in situ
up-regulation of proinflammatory cytokines, which is due to the
injection procedure and not to the IFN- production because it was
found in both d120:IFN-:LacZ- and
d120:LacZ-treated mice. MHC class II expression in the CNS
of IFN--treated mice indicates that IFN- gene delivery
induces CSF production of IFN- that, in turn, enters the CNS in a
bioactive form via the transependymal and transpial routes.
These results are in agreement with those previously found in rhesus
monkeys using the same delivery technique and the same backbone vector
(19).
IFN- gene delivery within the CNS before or after
EAE onset modifies the disease course but does not induce any
peripheral immunological effect
None of the mice injected with the d120:IFN-:\E
LacZ vector or with the d120:LacZ control vector,
before or after disease onset, died during the treatment or the
follow-up period, lasting 60 days postimmunization (p.i.).
Mice injected with the d120:IFN-:LacZ vector before EAE
onset (days 0 and 7 p.i.) showed an earlier disease onset
(11.2 ± 1.1 days p.i.), a milder disease course, and, in 5 of 6
(83%) mice, a complete recovery from the disease occurring between
days 22 and 28 p.i. (Fig. 3A, Table I). d120:LacZ control
vector-injected mice developed the disease later (19.6 ± 1.3 day
p.i.), had a higher maximum EAE score, and never recovered from the
disease (Fig. 3A, Table I). Untreated mice showed a EAE
course similar to that of d120:LacZ control vector-injected
mice (Fig. 3A, Table I).
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:LacZ vector or the
d120:LacZ control vector (Fig. 3
B, Table II). Mice injected with the
d120:IFN-:LacZ vector within 1 wk after EAE onset showed
a partial recovery from the disease at the time of sacrifice (from
1.6 ± 0.5 EAE score at disease onset to 1.2 ± 0.2 EAE score
23 days after the treatment) (Fig. 3B, Table II).
d120:LacZ control vector-injected mice never recovered after
the vector injection (Fig. 3B, Table II).
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:LacZ or the d120:LacZ control
vector. A comparable dose-dependent proliferation in response to the
immunizing Ag MOG35–55 (Fig. 4A) and to the polyclonal
stimulus Con A (Fig. 4B) was observed in LNC from
IFN--treated and control vector-treated mice. IFN- levels
secreted by LNC stimulated for 3 days with 10 µM of
MOG35–55 was not significantly different in mice
injected with the d120:IFN-:LacZ vector (6.3 ± 0.5
ng/ml) or with the d120:LacZ vector (11.8 ± 4.2 ng/ml;
p = 0.26).
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gene delivery within the CNS of EAE mice
increases the number of apoptotic cells and of TNFR1 expression in
CNS-infiltrating mononuclear cells
Apoptosis of CNS-infiltrating lymphocytes from EAE mice treated
with the IFN- or the control vector at days 0 and 7 p.i. were
quantified by staining with annexin V-FITC and PI on days 27 and
36 p.i. At day 27 p.i., the mice were recovering from the
initial acute phase of EAE, whereas at day 36 p.i. they had
already recovered (Fig. 3A). During the recovery phase, 85%
of gated lymphocytes were annexin V positive and PI negative in
IFN--treated mice, compared with 37.8% in control vector-treated
mice (Fig. 5A). After
recovery, 21.3% of gated lymphocytes from IFN--treated mice were
apoptotic compared with 8.7% (p = 0.05) in
controls (Fig. 5B). CNS-infiltrating mononuclear cells from
EAE mice treated with the IFN- or the control vector within 1 wk
after EAE onset were also analyzed for the expression of annexin V and
PI 27 days after vector injection. Again, 40.3% of gated lymphocytes
were annexin V positive and PI negative in IFN--treated mice,
compared with 18.6% in control vector-treated mice
(p = 0.03) (Fig. 5C). The presence
of apoptotic T cells among mononuclear cells infiltrating the CNS and
their persistence after disease recovery were confirmed in situ by
TUNEL analysis associated with anti-CD3 staining. Double-positive
apoptotic T cells were found within the subarachnoid space and the CNS
parenchyma in mice treated with the IFN--containing vector before
disease onset and sacrificed 60 days p.i. (Fig. 6, A and B).
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-treated
mice compared with control mice (9.66 vs 5.13 AU; p <
0.05). At day 36 p.i., the increased mRNA level of TNFR1 was still
measured in IFN--treated mice compared with control vector-treated
mice (6.59 vs 5.53 AU) although the difference was not any more
significant. The presence of TNFR1-expressing mononuclear cells
(among those infiltrating the CNS) was further confirmed in situ by
immunohistochemistry performed in mice treated with the
IFN--containing vector before disease onset and sacrificed 60 days
p.i. Positive cells were found within inflammatory infiltrates located
either in the spinal cord parenchyma or in the subarachnoid space (Fig. 6, C and D).
These results suggest that IFN- gene treatment induces
up-regulation of TNFR1 expression on blood-derived CNS-infiltrating
mononuclear cells that in turn die by apoptosis either within
subarachnoid spaces or in the CNS parenchyma.
Significant decrease of EAE pathology in IFN--treated mice
Neuropathological examination was performed 60 days p.i. only in
mice treated with IFN- vector or the control vector at days 0 and
7 p.i. The number of inflammatory perivascular infiltrates was
significantly (p = 0.03) reduced in
d120:IFN-:LacZ vector-treated (3.6 ±
0.5/mm2) compared with
d120:LacZ-treated control mice (5.4 ±
0.6/mm2). The significant reduction of
inflammatory infiltrates in IFN--treated mice was paralleled by
decreased percentages of demyelinated areas (0.3 ± 0.1 vs
3.4 ± 0.8/mm2; p = 0.0038)
and axonal loss (0.8 ± 0.2 vs 3.9 ±
0.8/mm2; p = 0.0019).
Because the i.c. delivery of the d120:LacZ:IFN- vector
determined IFN- production into the CSF compartment, we measured in
EAE mice treated with the IFN--containing vector the relative number
of T cells and macrophages infiltrating the spinal cord parenchyma from
the subarachnoid space or via the blood circulation. We found that the
number of macrophages was higher, although not significantly, in the
subarachnoid space (47.7 ± 15.9 cells/mm2)
compared with the CNS parenchyma (22.8 ± 4.9
cells/mm2). The same was observed for
CD3+ cells (CNS parenchyma = 33.6 ±
6.9 cells/mm2; subarachnoid space =
41.0 ± 6.9 cells/mm2). This indicates that
during EAE a substantial proportion of blood-derived CNS-infiltrating
mononuclear cells enters the CNS via the CSF, where our gene delivery
technique determines IFN- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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therapy showed a dramatic reduction of CNS inflammatory lesions, but
the therapy had little or no effect on neurological deterioration and
accumulation of demyelinating lesions (6). Treatment of MS
patients with anti-inflammatory molecules such as TGF-, TNFR1,
cladribine, glatiramer acetate, and Campath-1 failed to achieve
satisfactory therapeutic results and, to some extent, induced
paradoxical proinflammatory effects (4, 5, 6, 7, 8, 9). Here, we
further challenge the concept of an exclusive detrimental role of
inflammation in autoimmune demyelination by showing that the
administration in the CNS of EAE mice of IFN-, a prototypic
proinflammatory cytokine, determines a nearly complete recovery from a
chronic-progressive form of the disease.
IFN- was administered into the CNS by injecting, within the CSF
compartment of C57BL/6 mice, a nonreplicative HSV-1-derived vector
engineered with the mouse IFN- gene. This procedure
determines the exclusive infection by the vector of neuroectodermal
cells lining the CSF space (including those of the Virchow-Robin
spaces) and forming the blood-CSF barrier surrounding both the brain
and the spinal cord (i.e., ependymal, choroidal, and leptomeningeal
cells). Thus, the blood-CSF barrier prevents the vector from infecting
cells within the CNS parenchyma. Apart from a mild and transient
inflammatory reaction due to the injection procedure, which we
previously also observed in rhesus monkeys treated with the same
procedure and the same vectors (19), the vector itself
does not induce toxic or side effects in the CNS or in the periphery.
Vector-infected cells produced IFN- into the CSF for up to 3–4 wk
that entered the CNS parenchyma from the CSF compartment in its
bioactive form as indicated by the gradient of MHC class II
up-regulation observed in spinal cord cells of IFN--treated
mice.
The IFN- gene-containing vector administered before EAE
onset to mice immunized with MOG35–55 peptide
determined 1) a significant earlier onset of the disease; 2) a shorter
duration of the disease with a full recovery in almost 85% of mice;
and 3) a significant reduction of neuropathological signs of the
disease such as demyelination and axonal loss. EAE mice treated with
the IFN--containing vector within 1 wk after EAE onset showed a
substantial and significant recovery from the disease, which is
chronic-progressive in nature, within 30 days after the treatment. In
both cases, recovery from EAE was accompanied by a significant
increase in the percentage of apoptotic CNS-infiltrating
lymphocytes.
The protective effect of IFN- that we observed in EAE is somehow
surprising, because this cytokine is usually considered detrimental in
experimental as well as in human inflammatory demyelination. MS
patients treated with systemic IFN- showed a dramatic worsening of
the disease (20). Clinical exacerbation in MS
patients is usually accompanied by up-regulation of IFN- production
either in the CSF or in the blood (21). The protective
effect of IFN- therapy in MS patients has been attributed, at least
in part, to down-regulation of IFN--producing mononuclear cells
(22). In EAE, IFN- directs trafficking of leukocytes to
the CNS by orchestrating chemokine production (23).
IFN- is produced in the CNS by encephalitogenic
CD4+ cells by day 7 after immunization, peaks at
day 20, and then wanes (24), and its overproduction is
usually associated with relapses (25). This may explain
the earlier EAE onset we have observed in mice treated with the
IFN- gene-containing vector. However, a protective effect
for IFN- has been proposed in EAE. An increased EAE morbidity rate
(from 20 to 80%) was observed in C57BL/6J mice immunized with spinal
cord homogenate and Bordetella pertussis when treated with
neutralizing mAb against IFN- (11). In mice lacking the
gene coding for the ligand-binding chain of the IFN-R, IFN- is
not essential for the generation or function of anti
MOG35–55 effector cells but does play an
important role in down-regulating EAE during both effector and
induction phases (26). IFN-R-/-
mice developed EAE with high morbidity and mortality (up to 80%)
(26). Furthermore, the lack of IFN- converted an
otherwise EAE-resistant mouse strain to become susceptible to disease
(27). Nevertheless, transgenic mice expressing IFN- in
myelinating oligodendrocytes showed incidence, severity, and
histopathology of EAE similar to nontransgenic controls
(28).
How can our results help in explaining the protective effect of IFN-
in EAE? The HSV-1-derived nonreplicative vector containing the
IFN- gene entered exclusively cells within the CSF space
forming the blood-CSF barrier, thus being unable to infect cells in the
CNS parenchyma. Productive infection of CSF-lining cells determined the
release into the CSF of the biologically active form of IFN- that,
in turn, diffused to the CNS parenchyma. CNS production of IFN- did
not induce any pathological effect or recruitment of blood-borne
mononuclear cells. In addition, the gene delivery protocol did not
affect the peripheral immune system, as indicated by the similar T cell
proliferation to polyclonal stimuli or to the immunizing Ag in
IFN--treated and control mice. The protective effect of the
IFN- gene CNS delivery in EAE thus appears to be due to
an immunoregulatory activity exerted by the cytokine exclusively on
effector cells in the CNS. Using RT-PCR as well as
immunohistochemistry, TNFR1 was found up-regulated in blood-derived
CNS-infiltrating cells either confined within the subarachnoid space or
located into the CNS parenchyma, from IFN--treated mice. The
increased transcription of TNFR1 was paralleled by an increased
percentage of apoptotic CNS-infiltrating lymphocytes. We favor the
hypothesis that IFN- might have determined TNFR1 up-regulation on
CNS-infiltrating cells, entering the CNS parenchyma either via the CSF
compartment or via the blood stream. IFN- gene delivery
determined CSF production of bioactive IFN- that could also reach
the CNS parenchyma. In addition, IFN- can reach the depths of the
CNS parenchyma via the extensions of the subarachnoid space along
blood-penetrating vessels, the Virchow-Robin spaces. The activity of
IFN- in the subarachnoid space as well as in the CNS parenchyma can
be relevant from a therapeutic point of view because half of
CNS-infiltrating T cells and macrophages used the CSF route to access
the CNS, whereas the remaining came from the bloodstream. TNF-
produced in the CNS during EAE, as we have previously shown in the same
EAE model used in the present study (29), might have then
determined cell death by triggering the TNFR1. Our results are
supported by recent findings showing that 1) IFN- is able to
up-regulate TNFR1 expression on mononuclear cells
(30, 31, 32), 2) TNF- can induce apoptosis of
CD4+ and CD8+ T cells in
vivo (33), and 3) the impairment of TNFR1 signaling but
not Fas/FasL signaling decreases T cell apoptosis in MOG-induced EAE
(34, 35). Evidence supporting a protective role of TNF in
EAE is also available. TNF- treatment dramatically reduces disease
severity in both TNF--/- mice and in
TNF-+/+ mice highly susceptible to MOG-induced
EAE (36). In addition, chronic TNF- expression in vivo
may suppress the effector phase of autoimmune responses in vivo such as
those sustaining the progression of systemic and organ-specific
autoimmune diseases (37). However, we cannot exclude that
IFN- might have induced T cell apoptosis through the NO-induced cell
surface overexpression of IFN-R chains, as previously shown in vitro
in malignant and normal T cells (38), because NO is
considered one of the major final effector pathways in inflammatory
demyelination (2).
In conclusion, our data further challenge the concept that CNS inflammation is invariably detrimental in EAE/MS, and we suggest the existence of a protective countermechanism induced by inflammation that tends to restore, soon after the pathogenic event, the immune-privileged status of the CNS. Our data also challenge the over-simplistic view of the sole use of anti-inflammatory molecules in the therapy of EAE/MS.
|
Acknowledgments |
|---|
ELISA and Vincenzo Barnaba for helpful discussion. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gianvito Martino, DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail address: g.martino{at}hsr.it
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; BBB, blood brain barrier; CSF, cerebrospinal fluid; AU, arbitrary units; p.i., post immunization; MRI, magnetic resonance imaging; i.c., intracisternal(ly); LNC, lymph node cells; PI, propidium iodide; p.i., postimmunization; -Gal, -galactosidase.
Received for publication March 16, 2001. Accepted for publication May 30, 2001.
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0.05). Mean values (±SE) of at least three mice per group are shown
and are expressed as mRNA fold increase over basal levels measured in
five naive untreated C57BL/6 mice. Mean (±SE) mRNA levels in the whole
brain of untreated mice were as follows: IFN-
) and control mice (
) restimulated in vitro with the
indicated concentration of Con A. The background cpm values were
subtracted (
cpm). Each curve (±SE) or bar (±SE) represents the
mean cpm of three mice from each group per time point pooled together.
One representative set of experiments (of three performed) is
shown.
