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,
Departments of
*
Molecular Biology and Biochemistry and
Anatomy and Neurobiology, and
Reeve-Irvine Research Center, University of California, Irvine, CA 92612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), and CC
chemokine ligand 5 (RANTES) are expressed during both acute and
chronic stages of disease suggesting a role for these molecules in
disease exacerbation. Previous studies have shown that during the acute
phase of infection, T lymphocytes are recruited into the CNS by the
chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice
with established demyelination were treated with antisera against these
two chemokines, and disease severity was assessed. Treatment with
anti-CXCL10 reduced CD4+ T lymphocyte and macrophage
invasion, diminished expression of IFN- and CC chemokine ligand 5,
inhibited progression of demyelination, and increased remyelination.
Anti-CXCL10 treatment also resulted in an impediment of clinical
disease progression that was characterized by a dramatic improvement in
neurological function. Treatment with antisera against CXCL9 was
without effect, demonstrating a critical role for CXCL10 in
inflammatory demyelination in this model. These findings document a
novel therapeutic strategy using Ab-mediated neutralization of a key
chemokine as a possible treatment for chronic human inflammatory
demyelinating diseases such as MS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inducible protein 10 kDa) and CXCL9 (monokine
induced by IFN-) (2, 3, 4, 5). These chemokines operate by
binding to the receptor CXCR3 on T lymphocytes and NK cells
(6, 7, 8). A recent study has shown that CXCL10 and CXCL9 are
present within the cerebral spinal fluid of MS patients during clinical
attacks (3). In addition, postmortem analyses of MS brains
reveal increased expression of CXCL10 and CXCL9 by astrocytes and
CXCR3-positive T lymphocytes within inflammatory lesions undergoing
demyelination (3, 4, 5). These observations have led to the
hypothesis that CXCL10 and/or CXCL9 are key chemoattractants for the
inflammatory cells that contribute to demyelination. The availability
of blocking Abs for particular chemokines provides an opportunity to
test their role in contributing to inflammatory cell invasion and
myelin destruction in animal models of MS.
Intracerebral infection of mice with mouse hepatitis virus (MHV), a
(+)-strand RNA virus that is a member of the Coronaviridae
family, triggers a vigorous immune response involving both T
lymphocytes and macrophages (9, 10, 11, 12). CXCL10 and CXCL9 have
important roles in attracting leukocytes into the CNS during the acute
stage of disease. Indeed, neutralization of either CXCL10 or CXCL9
activity through administration of specific antisera results in
increased viral titers within the brain that correlates with decreased
T lymphocyte (both CD4+ and
CD8+ subsets) infiltration into the CNS and
diminished IFN- expression (11, 12).
Following the acute phase of infection, virus persists within white matter tracts, and mice often develop a chronic demyelinating disease in which there is continued leukocyte infiltration into the spinal cord, extensive myelin stripping, and neurological impairment including partial-to-complete hindlimb paralysis (13, 14). Chronic CXCL10 expression is associated with demyelinating lesions in MHV-infected mice suggesting that CXCL10 contributes to the pathogenesis of demyelination by attracting leukocytes into the CNS. Given the important role of CXCL10 during the acute inflammatory stage of disease, the present study evaluated whether administration of anti-CXCL10 antisera to persistently infected mice with established inflammation and demyelination could alter the course of disease.
We demonstrate that treatment with anti-CXCL10 antisera during the chronic phase of MHV-induced CNS disease prevents progression of demyelination within spinal cord white matter tracts and improves neurological function in persistently infected mice. In contrast, administration of anti-CXCL9 antisera was ineffective. These results document the feasibility of treating demyelinating diseases with Abs to chemoattractant molecules and also provides strong evidence that CXCL10 is a key chemoattractant during chronic demyelination in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MHV strain J2.2-V.1 was provided by J. Fleming (University of
Wisconsin, Madison, WI) (15). Age-matched (5- to
7-wk old) male C57BL/6 mice (H-2b background)
were used for all studies (National Cancer Institute, Bethesda, MD).
Following anesthetization by inhalation of methoxyflurane
(Pitman-Moore, Washington Crossing, NJ), mice were injected
intracranially with 1000 PFU of MHV suspended in 30 µl of sterile
saline. Control (sham) animals were injected with 30 µl of sterile
saline alone. Animals were sacrificed at days 7, 12, 21, and 28
postinfection (p.i.), at which point brains and spinal cords were
removed for analysis in studies described. For determination of viral
burden within the CNS, one-half of each brain was used for plaque assay
on the DBT astrocytoma cell line as previously described (assay
sensitivity is 
100 PFU/g tissue (2.0 log10);
Refs. 10, 11, 12).
Ab preparation and treatment of mice
Rabbit antisera specific to either CXCL10 or CXCL9 have
been previously described (11, 12, 16). These reagents
effectively neutralize activity in vivo and do not cross-react with
each other or other known chemokines (11, 12, 16). Mice
were injected i.p. with 0.5 ml of Ab (0.5 mg/ml) on days 12, 14, 16,
and 19 p.i. and sacrificed at days 21 and 28 p.i. Clinical
scoring was as follows: 0, no abnormality; 1, limp tail; 2, waddling
gait and partial hindlimb weakness; 3, complete hindlimb paralysis; 4,
death (10). Treatments were coded and clinical scores
determined independently by three investigators. Treatment with
increasing amounts of either anti-CXCL9 or anti-CXCL10 (>0.5
ml) did not result in improved clinical or histologic disease. A
control group was treated with 0.5 ml of normal rabbit serum (NRS).
Mononuclear cell isolation and flow cytometry
A single cell suspension was obtained from brains of mice treated with anti-CXCL10, anti-CXCL9, or NRS at days 21 and 28 p.i., and FACS analysis was performed as previously described (10). Briefly, brains were removed and homogenized by grinding the tissue followed by mincing with a razor blade to obtain a single cell suspension. Cell suspensions were transferred to 15-ml conical tubes and Percoll (Pharmacia, Uppsala, Sweden) was added for a final concentration of 30%. One milliliter of 70% Percoll was underlaid, and the cells were centrifuged at 1300 x g for 30 min at 4°C. Cells were removed from the interface and washed twice. FITC-conjugated rat anti-mouse CD4, CD8 (BD PharMingen, San Diego, CA), and F4/80 (Serotec, Oxford, England) were used to detect infiltrating CD4+ and CD8+ T lymphocytes as well as macrophages, respectively. As a control, an isotype-matched FITC Ab was used. Cells were incubated with Abs for 30 min at 4°C, washed, fixed in 1% paraformaldehyde, and analyzed on a FACStar (BD Biosciences, Mountain View, CA). Data are presented as the percentage of positive cells within the gated population. Data represent between three and six mice per treatment group examined in two separate experiments.
CD4+ and CD8+ T lymphocyte enrichment
Mononuclear cells were obtained from the CNS of experimental mice at days 21 and 28 p.i. according to the method described above, and a single cell suspension was obtained and pooled (three mice per group at each time point). Cells were resuspended in 90 µl of degassed, Ca2+ and Mg+2-free PBS supplemented with 0.5% BSA (MACS buffer) and stained with magnetic Abs specific for either CD4 or CD8 (Miltenyi Biotec, Auburn, CA) at a 1/10 dilution for 15 min at 4°C. Cells were washed, centrifuged at 300 x g for 10 min, and resuspended in 500 µl of MACS buffer. The cell suspension was then applied to a MACS MS+ separation column (Miltenyi Biotec), washed three times, and eluted in 1 ml of MACS buffer. Flow cytometry confirmed >95% purity of T lymphocyte fractions. Total RNA was isolated from the T cell subsets and subjected to RT-PCR to evaluate CXCR3 expression.
RT-PCR
Total RNA was extracted from brains of
CD4+ and CD8+ T lymphocyte
subsets at defined times p.i. using TRIzol reagent (Life Technologies,
Carlsbad, CA) and reverse transcribed with the AMV reverse
transcriptase system (Promega, Madison, WI). PCR amplification
was performed on resulting cDNA with primers specific for either
IFN- (Clontech Laboratories, South San Francisco, CA);
CXCR3: forward 5'-GCGGCCGCAACTCTTCCATTGTGGGCAG, reverse
5'-GAATTCAAGGCCCCTCGATAGAAGTT; CC chemokine ligand
(CCL)5: forward 5'-GTCGACGCGGGTACCATGAAGATCTCT, reverse
5'-TCTAGAAACCCTCTATCCTAGCTCATCTC; or L32: forward
5'-AACGCTCAGCTCCTTGACAT, reverse
5'-AACCCAGAGGCATTGACAAC. PCR amplification was
performed on an automated PerkinElmer (Norwalk, CT) model 480 DNA
thermocycler with the following profile: step 1, initial denaturation
at 94°C for 45 s; step 2, annealing at 60°C for 45 s; and
step 3, extension at 72°C for 2 min. Steps 1–3 were repeated 29
times for a total of 30 cycles. For cDNA generated from
CD4+ or CD8+ T lymphocyte
subsets, a total of 35 reaction cycles were used. PCR products were
analyzed by agarose gel electrophoresis and ethidium bromide staining.
Sequence analysis of the PCR amplicons confirmed primer
specificity.
ELISA
Brains were removed from MHV-infected mice that were
administered anti-CXCL10, anti-CXCL9, or NRS at 21 days p.i.
and homogenized (PowerGen 125; Fisher Scientific, Pittsburgh, PA) in
PBS and centrifuged at 1300 x g for 10 min. Following
collection of the supernatant, CCL5 (RANTES) and IFN- protein levels
in brain were determined using the Quantikine M mouse RANTES and
IFN- immunoassay kits according to manufacturers specifications (R&D
Systems, Minneapolis, MN) (10, 11, 12).
Histology
Spinal cords were removed at 21 and 28 days p.i. and fixed by immersion overnight in 10% normal buffered formalin after which portions of tissue were embedded in paraffin or TAAB resin (Electron Microscopy Services, Fort Washington, PA). The severity of demyelination was determined by three methods: 1) Luxol fast blue (LFB) staining of spinal cords (8 µm sections) and analysis by light microscopy. Demyelination was scored as follows: 0, no demyelination: 1, mild inflammation accompanied by loss of myelin integrity; 2, moderate inflammation with increasing myelin damage; 3, numerous inflammatory lesions accompanied by significant increase in myelin stripping; and 4, intense areas of inflammation accompanied by numerous phagocytic cells engulfing myelin debris (10). Slides containing stained spinal cord sections were blinded and scored by three investigators. Scores were averaged and presented as average ± SEM. The data presented represent a minimum of six spinal cord sections per mouse with three to six mice per time point. 2) To determine the extent of demyelination and remyelination, the lesion-containing length of spinal cord was cut into 1-mm transverse blocks and processed so as to preserve the cranio-caudal sequence and orientation. The tissue blocks were rinsed in 0.1 M phosphate buffer, pH 7.4, for 30 min, postfixed in 2% OsO4, dehydrated in ascending alcohols, and embedded in TAAB resin. Thin sections (1 µM) were cut from each block, stained with alkaline toluidine blue, and examined by light microscopy. 3) Electron microscopic analysis. Blocks were trimmed then cut at 100 nm, mounted on copper grids, uranyl acetate and lead citrate stained, and viewed under a Hitachi (Tokyo, Japan) EM 600 electron microscope at 75 kV. In adult animals there is a relationship between axon circumference and myelin sheath thickness (number of lamellae) expressed by the g-ratio (axon diameter: total fiber diameter); in remyelination this relationship changes such that myelin sheaths are abnormally thin for the axons they surround (17). An abnormally thin myelin sheath, relative to axonal diameter, was the criterion for oligodendrocyte remyelination. Absence of a myelin sheath was the criterion for demyelination. In all cases, slides were blinded and read independently by three investigators.
Statistical analysis
Statistically significant differences between groups of mice
were determined by t test using Sigma-Stat 2.0
software (Jandel, Chicago, IL), and p values of
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracranial infection of C57BL/6 mice with MHV results in an
acute encephalomyelitis with virus replicating primarily within glial
cells. Plaque assays document increased viral titers in the brain at
day 7 p.i. (3.9 ± 0.2 log10 PFU/g
tissue; n = 4), but by day 12 p.i., infectious
virus is not detected (<2 Log10 PFU/g tissue;
n = 4). The majority of animals survive the acute stage
of disease and develop a demyelinating disease characterized by
persistence of viral RNA and Ag within white matter tracts accompanied
by mononuclear cell infiltration and myelin destruction (10, 13, 15, 18, 19, 20). By day 12 p.i., animals developed clinical
disease characterized by awkward movement that progressed to
partial-to-complete hindlimb paralysis (Fig. 1
). T lymphocytes and activated
macrophage/microglia (as determined by F4/80 Ag expression) are present
within the CNS at this time, and demyelinating lesions are readily
detected (Table I).
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, Table I). Treatment with anti-CXCL10 resulted in a significant
reduction in clinical disease severity beginning at 15 days p.i. that
lasted until day 21 p.i. (p < 0.001)
(Fig. 1). During this time, mice treated with either anti-CXCL9 or
NRS exhibited hindlimb paralysis and limited mobility, whereas
anti-CXCL10-treated mice displayed almost complete restoration of
motor skills. Removal of anti-CXCL10 treatment (day 20 p.i.)
resulted in an increase in clinical disease severity such that by day
28 there were no differences between any experimental groups (Fig. 1). Treatment with anti-CXCL10 reduces T lymphocyte and macrophage infiltration into the CNS
Correlating with the decrease in clinical disease in mice treated
with anti-CXCL10 were FACS data demonstrating a significant
reduction in CD4+ T lymphocytes (58 and 55.2%
reduction, p < 0.02) and macrophage/microglia cells
(54.5 and 50% reduction, p < 0.01) present within the
CNS of these animals at day 21 p.i. as compared with T lymphocyte
and macrophage/microglia levels in anti-CXCL9- and NRS-treated
mice, respectively (Table I). Treatment with CXCL9 antisera did not
significantly reduce either CD4+ T lymphocyte or
macrophage/microglial cell entry into the CNS of infected mice (Table I). Although anti-CXCL10 and anti-CXCL9 treatment resulted in
an 25% decrease in CD8+ T lymphocyte levels
as compared with NRS-treated mice, this difference was not significant
(Table I). Activated T lymphocytes (both CD4+ and
CD8+ subsets) infiltrating into the CNS of
MHV-infected mice express CXCR3, the receptor for CXCL10 and CXCL9
(12). In an attempt to determine whether reduced T
lymphocyte infiltration into the CNS of anti-CXCL10-treated mice
correlated with reduced expression of CXCR3, RT-PCR was performed on
RNA isolated from either CD4+ or
CD8+ T lymphocyte subsets at defined times p.i.
Such analysis revealed a pronounced decrease in the level of CXCR3 mRNA
expression in CD4+ T lymphocytes isolated from
mice treated with anti-CXCL10 at day 21 p.i. when compared
with mice treated with NRS (Fig. 2A). In contrast, expression
of CXCR3 mRNA in CD8+ T lymphocytes was
comparable in mice treated with either anti-CXCL10 or NRS at the
same time point, supporting FACS data demonstrating similar levels of
CD8+ T lymphocyte infiltration at 21 days p.i.
(Fig. 2A). Interestingly, CXCR3 mRNA expression in
CD4+ T lymphocytes isolated from NRS-treated mice
at 21 days p.i. was markedly increased when compared with CXCR3 mRNA
expression in CD8+ T lymphocytes isolated from
either anti-CXCL10- or NRS-treated mice, suggesting differential
expression of this chemokine receptor on T lymphocyte subsets during
the chronic stage of MHV infection. Removal of anti-CXCL10
treatment (day 20 p.i.) resulted in an increase in the severity of
clinical disease symptoms that correlated with the return of
inflammatory CD4+ T lymphocytes and activated
macrophage/microglia into the CNS (Fig. 1 and Table I). Corresponding
with the return of inflammation within the CNS of mice formerly treated
with anti-CXCL10 is the increased expression of CXCR3 mRNA in the
CD4+ T lymphocyte subset at 28 days p.i. as
compared with levels at 21 days p.i. (Fig. 2A).
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Treatment with anti-CXCL10 reduces IFN- and CCL5 expression
Activated T lymphocytes obtained from the CNS of MHV-infected mice
during chronic disease express IFN- (21, 22). In
addition, CCL5 expression is associated with demyelinating lesions in
persistently infected mice, and expression of this chemokine has been
shown to contribute to demyelination by attracting macrophages into the
CNS (10). Analysis of CCL5 and IFN- expression at day
21 p.i. revealed decreased levels (as assessed by ELISA and
RT-PCR) in mice treated with anti-CXCL10 when compared with control
animals (Fig. 2, B and C). These data support
FACS analysis demonstrating the pronounced decrease in T lymphocyte
entry into the CNS as this population of cells is thought to produce or
influence the expression of CCL5 and IFN- in MHV-infected mice
(10, 12, 23). Moreover, the decreased macrophage
infiltration into the CNS of anti-CXCL10-treated mice can be
explained as a result of lowered levels of CCL5 (10).
Analysis of IFN- and CCL5 mRNA levels within the CNS at day 28
p.i. revealed comparable levels for both transcripts in mice formerly
treated with anti-CXCL10 and NRS (Fig. 2D). The
increased expression of both IFN- and CCL5 correlated with the
return of T lymphocytes and macrophages into the CNS as well as
clinical disease.
Treatment with anti-CXCL10 reduces the extent of myelin destruction and results in myelin repair
The fact that anti-CXCL10 treatment resulted in reduced
clinical disease severity and leukocyte infiltration into the CNS
suggested that demyelination may be affected as T lymphocytes and
macrophages are considered important contributors to demyelination in
MHV-infected mice (10, 23, 24). Indeed, LFB staining of
spinal cords indicated that mice treated with anti-CXCL10 exhibited
a marked reduction in the numbers of inflammatory foci present within
white matter tracts; this was accompanied by a significant reduction in
the severity of demyelination (p < 0.001;
0.8 ± 0.3, n = 6) when compared with either NRS-
(2.8 ± 0.1, n = 6) or anti-CXCL9 (3.3 ±
0.1, n = 4)-treated mice (Fig. 3 and Table I). Furthermore, additional
analysis of demyelination by toluidine blue staining of spinal cord
sections revealed that mice treated with NRS displayed numerous
inflammatory foci and robust demyelination throughout the ventral,
lateral, and dorsal columns (Fig. 4, B and D). In contrast, demyelination was limited
to the ventral column in mice treated with anti-CXCL10 supporting
the observation that progression of disease is impeded (Fig. 4, A and C). Removal of anti-CXCL10 treatment
correlated with a marked increase in the severity of demyelination such
that there were no appreciable differences between any of the
experimental groups by day 28 p.i. (Table I).
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, 80.1 ±
5.1% (n = 4 spinal cords) of the axons within the
ventral column lesion in anti-CXCL10-treated mice were surrounded by a
thin myelin sheath, characteristic of remyelination (Fig. 4E). In contrast, only 4 ± 0.71% (n =
3 spinal cords) of the axons within the ventral column lesion in
NRS-treated mice showed evidence of remyelination, the majority being
demyelinated (Fig. 4F). The relative failure of
remyelination in control mice may be due to the ongoing demyelination,
a conclusion that is supported by the increased number of T lymphocytes
and macrophages, as well as the increased levels of CCL5 and IFN- in
this treatment group. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression (11, 12).
These studies indicate that both CXCL9 and CXCL10 serve as important
sentinel molecules in host defense following MHV infection and
contribute to a protective Th1-mediated response. During chronic
disease, CXCL10 expression is primarily associated with demyelinating
lesions in MHV-infected mice suggesting an important role in
contributing to disease by attracting leukocytes into the CNS
(18). In addition, recent studies have indicated that T
lymphocytes are important contributors to demyelination in MHV-infected
mice (10, 23). Therefore, the present study was undertaken
to determine whether chronic expression of CXCL10 contributes to the
pathogenesis of MHV-induced demyelination by attracting T lymphocytes
into the CNS.
The data presented clearly indicate that administration of
anti-CXCL10 to MHV-infected mice with established demyelination
resulted in 1) an improvement in clinical disease, 2) diminished
infiltration of T lymphocytes and activated macrophage/microglia into
the CNS, 3) reduced expression of IFN- and CCL5, 4) suppression of
ongoing demyelination, and 5) a dramatically increased number of
remyelinated axons within lesions. These beneficial effects were fully
reversed when anti-CXCL10 treatment was discontinued. Specifically,
cessation of anti-CXCL10 delivery resulted in 1) the restoration of
clinical disease, 2) the return of T lymphocytes and
macrophage/microglia within the CNS, 3) comparable levels of IFN-
and CCL5 mRNA expression, and 4) robust demyelination. Taken together,
the results presented provide the first clear evidence that CXCL10 is
an important contributor to the pathogenesis of demyelination in the
MHV model system by regulating inflammatory cell infiltration into the
CNS during the chronic phase of disease. In addition, these studies
illustrate the delicate balance that exits between chemokine expression
as it relates to host defense and disease development. Specifically,
early expression of CXCL10 is beneficial as it serves to attract
antiviral T lymphocytes into the CNS that participate in elimination of
virus (12). Paradoxically, persistent infection of mice
leads to chronic expression of CXCL10 that results in T lymphocyte
infiltration into the CNS. Once present, activated T lymphocytes
influence the expression of CCL5, which has been shown to contribute to
disease pathogenesis, through attraction of additional T lymphocytes
and macrophages, that ultimately results in myelin destruction
(10, 23). Importantly, the current study supports and
extends earlier work demonstrating that Ab-mediated targeting of CXCL10
results in a modulation in the severity of neuroinflammation and
disease development (12).
Neutralization of CXCL10 activity resulted in a dramatic
improvement in the clinical status of treated animals (Fig. 1). Within
3 days of treatment, mice treated with anti-CXCL10 displayed a
significant improvement in clinical disease as compared with mice
treated with either anti-CXCL9 or NRS. Upon cessation of
anti-CXCL10 treatment at day 20 p.i., anti-CXCL10 animals
continued to display only minor signs of clinical disease as compared
with the other experimental groups. However, by day 22 p.i., mice
formerly treated with anti-CXCL10 began to show signs of worsening
disease, e.g., limited mobility and paralysis such that at day 28
p.i. there were no differences in clinical disease severity between
experimental groups. Continued treatment of mice with anti-CXCL10
antisera until day 28 p.i. prolongs the reduction in clinical and
histologic disease only slightly (data not shown). The limited period
that anti-CXCL10 treatment has on MHV-induced CNS disease most
likely reflects accelerated decay of the rabbit polyclonal antisera
resulting from degradation and/or elimination of the reagent by the
host immune response rather than a transient role for CXCL10 in
contributing to chronic demyelination. Furthermore, previous studies
have shown almost exclusive expression of CXCL10 within and around
demyelinating lesions of mice persistently infected with MHV as late as
35 days p.i., indicating a prominent role for CXCL10 in contributing to
demyelination throughout the chronic stage of disease
(18). Therefore, we believe that CXCL10 does not influence
leukocyte infiltration and demyelination for only a limited time but
throughout the course of disease.
Anti-CXCL10 treatment had a pronounced effect upon
CD4+ T lymphocyte recruitment (55% reduction at
day 21 p.i. as compared with control mice), whereas
CD8+ T lymphocyte infiltration (26% reduction at
day 21 p.i.) into the CNS was not significantly affected (Table I). The marked reduction in CD4+ T lymphocyte
infiltration in mice treated with anti-CXCL10 correlated with an
inhibition in the progression of demyelination. These data support and
extend previous studies indicating an important role for this
population of cells in amplifying the severity of demyelination in
MHV-infected mice (10, 23). We have previously proposed
that CD4+ T lymphocytes may accomplish this by
producing and/or influencing the expression of CCL5 that serves to
attract macrophages into the CNS (10). The data presented
in this report support these earlier observations in that the reduction
in CD4+ T lymphocyte infiltration into the CNS of
anti-CXCL10-treated mice correlated with reduced expression of
CCL5, which was accompanied by a dramatic decrease in
macrophage/microglia levels (Table I). Correspondingly, removal of
anti-CXCL10 treatment resulted in the return of
CD4+ T lymphocytes, increased expression of CCL5,
and increased numbers of macrophage/microglia as well as robust
demyelination. Collectively, these data indicate that CXCL10 promotes
demyelination by attracting CD4+ T lymphocytes
into the CNS that, in turn, accelerate disease through CCL5 expression
and macrophage infiltration.
Although CD8+ T lymphocyte infiltration into the
CNS was reduced in mice treated with anti-CXCL10, the effect was
not significant when compared with mice treated with either
anti-CXCL9 or NRS (Table I). These data were somewhat surprising in
light of our previous study that indicated that neutralization of
CXCL10 resulted in >75% decrease in both CD4+
and CD8+ T lymphocyte entry into the CNS during
acute disease (12). One possible mechanism contributing to
this observation is that CD8+ T lymphocytes do
not express similar levels of CXCR3 as compared with
CD4+ T lymphocytes during the chronic stage of
MHV infection. This theory is supported by the decreased level of CXCR3
mRNA expression observed in CD8+ T lymphocytes as
compared with expression in the CD4+ T lymphocyte
subset at both 21 and 28 days p.i. (Fig. 2A). In addition,
it is also possible that CD8+ lymphocytes may be
responding to different chemotactic signals during the chronic stage of
disease.
An important observation was the pronounced suppression of ongoing demyelination accompanied by improved neurological function in anti-CXCL10-treated mice. The gross increase in remyelination suggests that diminished effector cell recruitment, e.g., CD4+ T lymphocyte and macrophages, into the CNS halts disease progression and repair may be initiated. However, the mechanism(s) by which repair is undertaken are currently unknown at this time. It is possible that lowered numbers of CD4+ T lymphocytes and macrophages result in decreased levels of potentially cytotoxic factors that allow for increased expression of myelin-encoding genes (30, 31). Alternatively, oligodendrocyte progenitor cells, which are present within the adult CNS and have been shown to participate in remyelination, may be attracted to damaged areas within the spinal cord in the absence of CXCL10 activity (32, 33, 34). Although we do not know whether myelin repair in the anti-CXCL10-treated mice is associated with functional recovery from conduction block, other studies have demonstrated that conduction velocity and frequency response properties of axons are restored to near normal values following remyelination (35).
In summary, the data presented offer new insight into the functional significance of CXCL10 expression during MHV-induced demyelinating disease. Extrapolation of these data to human neuroinflammatory diseases such as MS indicate that therapies designed at targeting CXCL10 should be considered as a viable treatment strategy for reducing the severity of neuroinflammation and demyelination.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Molecular Biology and Biochemistry, University of California, 3205 Biological Sciences II, Irvine, CA 92697-3900. E-mail address: tlane{at}uci.edu
3 Abbreviations used in this paper: MS, multiple sclerosis; MHV, mouse hepatitis virus; NRS, normal rabbit serum; p.i., postinfection; CXCL, CXC chemokine ligand; CCL, CC chemokine ligand; LFB, Luxol fast blue.
Received for publication March 13, 2001. Accepted for publication July 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible chemokines IP-10 and Mig and their receptor, CXCR3, in multiple sclerosis lesions. Neuropathol. Appl. Neurobiol. 26:133.[Medline]
and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873.) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system. J. Immunol. 166:1790. and 
chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160:970.. Nat. Med. 9:1037.
R during early remyelination. J. Neurobiol. 37:413.[Medline]
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J. Miu, A. J. Mitchell, M. Muller, S. L. Carter, P. M. Manders, J. A. McQuillan, B. M. Saunders, H. J. Ball, B. Lu, I. L. Campbell, et al. Chemokine Gene Expression during Fatal Murine Cerebral Malaria and Protection Due to CXCR3 Deficiency J. Immunol., January 15, 2008; 180(2): 1217 - 1230. [Abstract] [Full Text] [PDF]
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