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-Inducible Protein-10) Control of Encephalitogenic CD4+ T Cell Accumulation in the Central Nervous System During Experimental Autoimmune Encephalomyelitis1
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Department of Pathology, Immunobiology Center, Robert H. Lurie Cancer Center, and Institute for Neuroscience, Northwestern University Medical School, Chicago, IL 60611;
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and
Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10) in the
pathogenesis of EAE. Production of CXCL10 in the CNS correlated with
the development of clinical disease. Administration of anti-CXCL10
decreased clinical and histological disease incidence, severity, as
well as infiltration of mononuclear cells into the CNS. Anti-CXCL10
specifically decreased the accumulation of encephalitogenic
PLP139–151 Ag-specific CD4+ T cells in the CNS
compared with control-treated animals. Anti-CXCL10 administration did
not affect the activation of encephalitogenic T cells as measured by
Ag-specific proliferation and the ability to adoptively transfer EAE.
These results demonstrate an important role for the CXC chemokine
CXCL10 in the recruitment and accumulation of inflammatory mononuclear
cells during the pathogenesis of EAE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An essential step during the pathogenesis of tissue-specific
inflammatory diseases is the chemokine-induced recruitment of
leukocytes. Chemotactic cytokines (chemokines) are molecules that
induce leukocyte accumulation in tissue sites of inflammation (9, 10) that can be divided into four highly conserved, but distinct
families: the CXC, CC, C, and CX3C families,
based on the position of the cysteines in the amino terminus portion of
the molecule (11, 12). CC chemokine family members have
been implicated as candidates in the immunopathology of EAE as T cell
production of CCL3 (macrophage-inflammatory protein-1
(MIP-1))
and CCL1 (T cell activation protein-3 (TCA-3)) were shown to be
associated with T cell clones that were able to induce the adoptive
transfer of EAE (13). MIP-1, CCL2 (monocyte chemotactic
protein-1 (MCP-1)), and CXCL10 (IFN--inducible protein-10 (IP-10))
expression in the CNS has been associated with acute disease symptoms
in both rat (14) and murine EAE models
(15, 16, 17, 18). Functional significance of MIP-1 and MCP-1 in
EAE pathogenesis was demonstrated using neutralizing Abs to inhibit
disease onset with the administration of anti-MIP-1
(17) or ameliorate relapsing disease severity with the
administration of anti-MCP-1 (18).
The important role chemokine receptors have in recruiting cells into the CNS during the induction of clinical EAE has recently been identified. CCR2-/- mice were shown to be protected from clinical EAE in an active disease model (19, 20). CCR2 was shown to be important for the recruitment of peripheral macrophages to the CNS in a series of adoptive transfer experiments using CCR2-/- or wild-type CD4+ T cells transferred to wild-type or CCR2-/- recipients (19). In the absence of CCR2, CD4+ T cells from wild-type animals were able to induce clinical disease (19). However, when wild-type CD4+ T cells were transferred to CCR2-/- recipients, clinical disease was not observed, due to a significant decrease of peripheral macrophages accumulating in the CNS (19). CCR1-/- mice have also been shown to have reduced clinical EAE in an active disease model (21), demonstrating partial disease protection. These findings demonstrate an important role for chemokine receptor expression and CNS mononuclear cell infiltration.
In the present study, we investigated the potential role IP-10 plays
during Ag-specific T cell migration/accumulation in the pathogenesis of
an organ-specific, T cell-mediated autoimmune disease. IP-10 is a
member of a subset of chemokines including CXCL11 (IFN-inducible T cell
-chemoattractant (I-TAC)) and CXCL9 (monokine induced by IFN-
(Mig)), which are regulated by the expression of IFN-
(22, 23, 24). CXCR3, the receptor for these chemokines, is
expressed on a subset of circulating T cells (25);
however, expression of CXCR3 and IP-10 chemotaxis are increased
dramatically after T cell activation (26), and CXCR3
expression has been shown to be associated with Th1 cell lines and
clones (27, 28). CXCR3 expression has also been detected
on infiltrating monocytes within demyelinating MS brain lesions where
high levels of IP-10 are expressed (29). EAE is a
CD4+ Th1-mediated demyelinating disease of the
CNS, we therefore investigated the role IP-10 plays in the recruitment
of CD4+ Th1-autoreactive cells into the CNS
during the pathogenesis of autoimmune encephalomyelitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL mice (H-2s) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Congenic SJL.Thy1a mice were received from H. Tse (30) (Wayne State University, Detroit, MI) and subsequently bred and maintained at Northwestern University (Chicago, IL). Mice were 6–7 wk old at the initiation of the experiment and were maintained on standard laboratory chow and water ad libitum. Animals were housed under specific pathogen-free, barrier facility conditions. Animal care was provided in accordance with the Northwestern University and National Institutes of Health guidelines.
Antigens
PLP139–151 (HSLGKWLGHPDKF) was purchased from Peptides International (Louisville, KY). The amino acid composition was verified by mass spectrometry, and purity (>98%) was assessed by HPLC.
Priming of donor lymphocytes, cell culture, and transfer of EAE
Donor congenic SJL.Thy1a or normal SJL mice were primed by s.c. immunization with 50 µg of PLP139–151 emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco, Detroit, MI). Seven days later, draining lymph node cells were pooled and cultured in vitro for 72 h in complete DMEM (Life Technologies, Grand Island, NY) containing 5 x 10-5 M 2-ME (Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 0.1 M nonessential amino acids (Life Technologies), and 10% FCS (HyClone, Logan, UT) at 6 x 106 cells/ml in the presence of 50 µg/ml PLP139–151. Cells were incubated at 37°C in a humidified atmosphere containing 7.5% CO2. The cells were harvested after 72 h culture, washed, and 5 x 106 viable T cell blasts were transferred i.v. to normal SJL recipients. Subsequent to cell transfer mice were evaluated for the development of EAE.
Clinical evaluation
Adoptive R-EAE was induced by the transfer of 5 x 106 in vitro-stimulated, PLP139–151-specific T cell blasts from PLP139–151 peptide-primed mice. Individual animals were observed daily and graded according to their clinical severity as follows: grade 0, no abnormality; grade 1, limp tail; grade 2, limp tail and hind limb weakness (waddling gait); grade 3, partial hind limb paralysis; grade 4, complete hind limb paralysis; grade 5, death.
Isolation of CNS-infiltrating mononuclear cells, lymph node cells, and splenocytes
Mice were anesthetized with methoxyflurane (Pitman-Moore,
Mundelein, IL) and perfused through the left ventricle with 
60 ml of
PBS. Spinal cords were extruded by flushing the vertebral canal with
PBS and rinsed in PBS. Lymph nodes and spleens were removed from the
same mice and placed in HBSS. Tissues were forced through 100-mesh
stainless steel screens to yield a single-cell suspension. RBC in the
spleen preparations were lysed by hypotonic shock in
Tris-NH4Cl (pH 7.3), and the cells were washed
and resuspended in HBSS. CNS mononuclear cells were isolated by
centrifugation (500 x g) at 24°C on a 30–70%
discontinuous Percoll (Pharmacia, Piscataway, NJ) gradient. Cells were
collected from the interface, washed in HBSS, and resuspended in
isotonic-buffered saline (IBS) (Baxter Diagnostics, McGaw Park, IL)
containing 0.1% NaN3 and 0.2% BSA (Sigma, St.
Louis, MO).
Antibodies
mAbs to murine Thy1a (CD90.1; HIS51), Thy1b (CD90.2; 53-2.1),
CD4 (RM4-5), CD8a (Ly-2), CD19 (1D3), CD45 (Ly-5), and CD16/32 (2.4G2,
anti-mouse FcRII/III) were purchased from BD PharMingen (San
Diego, CA). mAbs to murine F4/80 were purchased from Caltag
Laboratories (Burlingame, CA). Isotype control Abs were purchased from
BD PharMingen. Rabbit anti-human polyclonal IP-10 Ab was prepared
by multiple-site immunization of New Zealand White rabbits with
recombinant human IP-10 (PeproTech, Rocky Hill, NJ) in CFA (31, 32). The specificity of the IP-10 anti-serum recognized both
murine and human forms of IP-10 and was confirmed by ELISA and Western
blot analysis (31, 32). Polyclonal anti-IP-10 Abs were
titered by direct ELISA (106 titer), and
specificity was verified by the failure to cross react with any of the
following human or mouse CC or CXC family chemokines: growth-related
oncogene (GRO)-, GRO-
, GRO-, IL-8, epithelial cell-derived
neutrophil-activating factor 78 amino acids, Mig, I-TAC,
neutrophil-activating protein-2, granulocyte chemoattractant protein-2,
mouse MIP-2, mouse KC, mouse Mig, RANTES, mouse C10, eotaxin,
macrophage-derived chemokine, thymus- and activation-related chemokine,
human and mouse MIP-1, human and mouse MIP-1, and human and mouse
MCP-1 (31, 32). The IgG portion of the serum was purified
over a protein A column and used in a sandwich ELISA while whole serum
(0.5 ml) was used for in vivo treatments to block IP-10
(31). Neutralizing hamster anti-murine IP-10 mAb was
generated by immunizing Armenian hamsters with recombinant
Escherichia coli-produced murine IP-10 in CFA as previously
described (33). mAbs were tested for their specificity
using available purified mouse chemokines, including Mig, I-TAC,
MIP-1, MIP-1, stromal cell-derived factor-1, KC, TCA-3, RANTES,
eotaxin, MCP-1, MCP-3, and MCP-5 in a direct ELISA and immunoblot assay
(33). The ability to inhibit IP-10-induced chemotaxis was
demonstrated in vitro and in vivo using this mAb (33).
Flow cytometry
Cells (0.5–1 x 106) were incubated
with anti-mouse FcRII/III for 15 min at 4°C in to block
Fc-mediated binding. Cells were washed in IBS followed by incubation
with specific Abs CD90.1, CD90.2, CD4, CD8a, CD19, CD45, or F4/80 at a
predetermined optimal concentration for 15 min at 4°C. As a control,
parallel populations of cells were incubated in the presence of
isotype-matched control Abs. Cells were washed and resuspended in 0.5
ml IBS. Data collection and analysis were performed on a FACSCalibur
(Becton Dickinson, San Jose, CA) flow cytometer using CellQuest
software with 5 x 104 events/analysis.
Cell sorting
Splenocytes and infiltrating mononuclear cells were isolated
from the CNS as described above and were incubated with Fc Block
(CD16/32 clone 2.4G2; BD PharMingen) at 1 x
106 cells/ml for 15 min at 4°C in IBS to block
Fc-mediated binding. Cells were washed in IBS followed by incubation
with directly conjugated CD90.1 FITC and CD4 PE mAbs for 15 min at
4°C in the dark at a predetermined optimal concentration. Cells were
washed twice in IBS and resuspended in IBS at final concentration of
1 x 106 cells/ml for the CNS and 1 x
107 cells/ml for the spleen cells.
CD4+CD90.1+ and
CD4+CD90.1- cells were
sorted from the CNS and spleen cell preparations using an EPICS Elite
cell sorter (Beckman Coulter, Fullerton, CA). Cells were collected in
HBSS supplemented with 2% FCS, pelleted, and lysed in TRIzol (Life
Technologies) and stored at -70°C for RNA isolation. Following cell
sorting, an aliquot of cells from each tube was analyzed for percent
purity by flow cytometry. All sorted populations had 
98% purity.
RT-PCR
Total RNA was isolated from the sorted cells using TRIzol (Life Technologies) following the manufacturer’s suggested protocol. Total RNA was quantitated by measuring A260/A280 absorbance. Equal amounts of total RNA were used for the synthesis of cDNA using the first-strand cDNA synthesis kit from Clontech Laboratories (Palo Alto, CA) following the manufacturer’s suggested protocol. PCR was performed using a Perkin-Elmer 9600 thermocycler (Norwalk, CT). Chemokine receptor primers were designed using software provided by National Center for Biotechnology Information and synthesized by Life Technologies. PCR conditions for G3PDH and CXCR3 were as follows: 94°C for 3 min, followed by 40 cycles of 30 s at 94°C, 30 s at 62°C, and 1 min at 72°C, with a final extension at 72°C for 3 min. Primer sequences used in this study: CXCR3 sense, 5'-GAACGTCAAGTGCTAGATGCCTCG-3'; antisense, 5'-GTACACGCAGAGCAGTGCG-3'; and G3PDH sense, 5'-ACCACAGTCCATGCCATCAC-3'; antisense, 5'-TCCACCACCCTGTTGCTGTA-3'.
Histology
Histological evaluation was performed on representative mice
from each experimental group. Mice were anesthetized with
methoxyflurane (Pitman-Moore) and perfused through the left ventricle
with 60 ml of PBS. Spinal cords were extruded by flushing the
vertebral canal with PBS. The most caudal 1 cm of the lumbar spinal
cord was fixed in a phosphate-buffered 10% formalin solution and
embedded in paraffin. Twelve 10-µm sections from each animal were
stained with hematoxylin and eosin and examined for the presence of
mononuclear cell infiltration. Histological scores were determined
using the following scale: 0, no mononuclear cell infiltration; 1, 1–5
perivascular lesions per section with parenchymal infiltration; 2,
5–10 perivascular lesions per section with parenchymal infiltration;
and 3, >10 perivascular lesions per section with extensive parenchymal
infiltration. The mean histological score ± SD was calculated for
each group. Representative photomicrographs were taken using a Nikon
microscope (Fryer Company, Huntley, IL) equipped with a SPOT digital
camera (Diagnostic Instruments, Sterling Heights, MI). Images were
created using Metamorph Meta Imaging Series 4.5 software (Universal
Imaging Corporation, West Chester, PA) and printed with a Fujix
pictrography 3000 (Fuji Photo Film USA, Elmsford, NY).
Cytokine and chemokine ELISA
Assessment of cytokine production was determined from lymph node
culture supernatants harvested following 48 h stimulation with 0,
0.5, 5, or 50 µM PLP139–151 peptide. The
supernatants were tested for the presence of IL-2, IL-4, and IFN- by
commercial ELISA kits (Endogen, Cambridge, MA). Assessment of IP-10 was
quantitated from tissue samples using previously described
noncommercial ELISA (31). Briefly, spinal cord samples
were homogenized in 1 ml PBS and clarified by centrifugation (400
x g) for 10 min. Flat-bottom microtiter plates (Nunc,
Naperville, IL) were coated with capture Ab diluted to 3.2 µg/ml in
borate-buffered saline coating buffer and blocked with 2% BSA (Sigma)
in PBS for 1 h at room temperature, and samples were subsequently
added in triplicate and incubated overnight at room temperature.
Biotinylated goat anti-rabbit detection Ab was added, and the
plates were incubated for an additional 1 h at room temperature.
The plate was developed using streptavidin-peroxidase (Zymed, South San
Francisco, CA) and o-phenylenediamine substrate (Sigma), and
absorbance was read at 490 nm using a Vmax
kinetic microplate reader (Molecular Devices, Sunnyvale, CA). Standard
curves for the individual cytokines and chemokines were generated using
a series of dilutions of purified recombinant protein (R&D Systems,
Minneapolis, MN). Chemokine levels in spinal cords were quantitated by
comparison to the standard curves and expressed as nanograms per
milliliter. The individual chemokine Abs used in the ELISA are specific
and do not cross-react with any other chemokine as described above. The
detection limit of the cytokine ELISA kits was as follows: IL-2, 15.6
pg/ml, IL-4, 31.3 pg/ml, and IFN-, 48.8 pg/ml. The detection limit
for the chemokine IP-10 was 312.5 pg/ml.
In vitro T cell proliferation assays
Spleen and lymph node cells were obtained from mice at the peak of acute clinical disease for the control-treated group. Tissues were forced through 100 mesh stainless steel screens to yield a single-cell suspension. RBC in the spleen preparations were lysed by hypotonic shock in Tris-NH4Cl (pH 7.3), and the cells were washed and resuspended in HBSS. Cells were cultured in 96-well microtiter plates (Corning-Costar, Acton, MA) at 5 x 106 viable cells/ml in DMEM (Life Technologies) containing 5 x 10-5 M 2-ME (Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 0.1 M nonessential amino acids (Life Technologies), and 5% FCS (HyClone, Logan, UT) in the presence of 0, 0.5, 5, and 50 µM PLP139–151. Cells were incubated at 37°C in a humidified atmosphere containing 7.5% CO2. The cells were pulsed with 1 µCi of [3H]TdR (ICN Radiochemicals, Irvine, CA) after 72 h, harvested after 96 h, and [3H]TdR uptake was detected using a Packard Topcount microplate scintillation counter (Packard Instruments, Meriden, CT). Results are presented as the mean ± SEM of triplicate wells.
Statistical analysis
Comparison of disease incidence were analyzed by
2 test, using Fisher’s exact probability
test. Statistical significance of cytokine levels, thymidine
incorporation, disease onset, and disease severity was analyzed using
Student’s t test for comparisons of two means. Values of
p 
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We wanted to determine whether there was a relationship between
CNS IP-10 protein production and development of clinical EAE. To
analyze this, we examined organ-specific chemokine protein production
by ELISA. EAE was induced in SJL mice by adoptive transfer of
PLP139–151-specific, in vitro-activated T cells.
Mice typically begin to show clinical signs of disease 5–14 days
following transfer of activated T cells. A kinetic analysis of IP-10
induction was performed on groups of mice 0, 2, 6, 8, 9, and 11 days
following transfer of encephalitogenic T cells. The spinal cords were
snap frozen in liquid nitrogen, homogenized in PBS, and analyzed for
the presence of IP-10 by ELISA. Fig. 1
demonstrates that increasing IP-10 production in the CNS precedes the
development of clinical disease symptoms as well as maintains high
levels during peak clinical disease.
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We next wanted to determine whether there was a correlation
between CNS IP-10 mRNA expression and development of clinical EAE. EAE
was induced in SJL mice by adoptive transfer of
PLP139–151-specific, in vitro-activated T cells.
A kinetic analysis of IP-10 mRNA expression was performed on groups of
mice 1, 2, 3, 4, and 9 days following transfer of encephalitogenic T
cells. Total RNA from the spinal cords was isolated and analyzed for
the presence of IP-10 by RT-PCR. Fig. 2A demonstrates IP-10 mRNA
expression in the CNS precedes the development of clinical disease
symptoms and increases at disease onset, day 9. From the observation
that IP-10 is produced in the CNS following EAE induction, we would
predict that T cells accumulating in the CNS following EAE induction
would express CXCR3, the receptor for IP-10. To assess this
possibility, EAE was induced in normal SJL (Thy1b) mice by the adoptive
transfer of congenic SJL.Thy1a
PLP139–151-specific T cell blasts. By inducing
disease with congenic Thy1a cells, we were able to track the
encephalitogenic CD4+ T cells in normal SJL
(Thy1b) recipients using specific mAbs (CD90.1 and 90.2, respectively).
At the peak of acute EAE, the CNS-derived mononuclear cells and
splenocytes were labeled with Abs to CD4 and CD90.1. Double-positive
CD4+CD90.1+ T cells
(encephalitogenic donor cells) and single-positive
CD4+CD90.1- T cells (host
derived) were sorted from these tissues as described in Materials
and Methods. Sorted cells were analyzed by RT-PCR for CXCR3 mRNA
expression. CXCR3 was expressed by both donor- and host-derived
CD4+ T cells isolated from the CNS during peak
acute clinical disease, day 12 post adoptive transfer (Fig. 2B). In contrast, CXCR3 expression was not found on either
CD4+CD90.1+
(encephalitogenic donor cells) or
CD4+CD90.1- (host-derived)
T cells from sorted splenic populations (Fig. 2B). Taken
together, these data demonstrate the presence of both IP-10 and its
receptor, CXCR3, in the CNS during the development of acute
EAE.
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To determine the biological significance of IP-10 expression
during acute EAE, we tested the ability of Ab against IP-10 to affect
adoptive EAE in recipient mice. Administration of 0.5 ml of
anti-IP-10 i.p. on days 0 and 2 relative to adoptive transfer of
encephalitogenic T cells significantly decreased clinical disease
compared with normal rabbit serum (NRS) control-treated animals (Fig. 3). All NRS control-treated recipients
developed severe EAE with a mean clinical score of 3.9, while only 2 of
6 (33%) of the recipients in the anti-IP-10-treated group
developed disease with delayed onset, p < 0.001, and a
significantly decreased mean clinical score of 0.8, p
< 0.0001. The results presented here demonstrate that neutralization
of IP-10 during disease initiation, but before the development of
clinical symptoms, significantly reduces the severity of acute EAE. In
a separate experiment, EAE was induced by the adoptive transfer of
PLP139–151-specific T cells, and the ability of
an independent hamster anti-murine IP-10 mAb to ameliorate clinical
disease was tested. The hamster Ig-treated control group developed
severe EAE (mean clinical score = 1.7; incidence = 14/14),
while the anti-mIP-10 mAb-treated group developed significantly
less (p < 0.003) severe disease (mean clinical
score = 1.0; incidence = 11/14). These data provide an
important confirmation of the biological role of IP-10 in the
pathogenesis of EAE using a mAb specific for murine IP-10. Taken
together, these results demonstrated that in vivo neutralization of
IP-10 before the onset of clinical EAE significantly reduced the
clinical disease severity.
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To determine the requirement for IP-10 recruitment of mononuclear
cell infiltration into the CNS, mice were examined for spinal cord
pathology. Longitudinal lumbar spinal cord sections from
anti-IP-10-treated mice and NRS control mice were prepared as
described in Materials and Methods and evaluated for the
extent of mononuclear cell infiltration of the meninges, perivascular
areas, and parenchyma by hematoxylin and eosin staining. It should be
emphasized that at the time of histological examination, the
anti-IP-10-treated mice showed no signs of clinical EAE, while the
NRS control mice showed severe clinical EAE (Fig. 3
). Spinal cord
sections from NRS control mice exhibited extensive meningeal,
perivascular, and parenchymal infiltrates (Fig. 4A). In contrast, spinal cord
sections from anti-IP-10-treated mice demonstrated no meningeal,
parenchymal, or perivascular cell infiltration (Fig. 4B).
These histological results were quantitated and as shown in Table I the anti-IP-10-treated mice lacked
CNS mononuclear cell infiltration and had a significantly decreased
number of lesions per section compared with the NRS control mice. These
results suggest that IP-10 is required for mononuclear cell
accumulation in the CNS.
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Administration of neutralizing IP-10 antisera results in a
significant reduction of clinical and histological EAE. We next
examined whether there was a change in mononuclear cell numbers in the
CNS and periphery as a result of anti-IP-10 treatment. When
control-treated mice showed peak clinical disease, CNS, lymph node, and
spleen mononuclear cells from control- and anti-IP-10-treated mice
were enumerated. The results shown in Fig. 5 demonstrate a decrease in total
mononuclear cells in the CNS from anti-IP-10-treated mice compared
with control-treated mice.
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Flow cytometric analysis of the CNS was performed to determine
whether anti-IP-10 treatment changed the cellular composition of
the mononuclear cell infiltrate. EAE was induced in normal SJL (Thy1b)
mice by the adoptive transfer of congenic SJL.Thy1a
PLP139–151-specific CD4+ T
cell blasts. The immunophenotyping results are shown in Table II and reveal that control mouse spinal
cord infiltrate was composed of T cells (CD4+ and
CD8+) and F4/80+ cells as
expected. Not only was there a decrease in the total number of CNS
mononuclear cells in the anti-IP-10-treated cords (20-fold,
Table II), but there was also a decrease in the percentage of T cells
(<1%, Table II). CD4 and CD90.1 staining allowed us to distinguish
between the disease-inducing
CD4+CD90.1+
(Thy1a+) cells and the host-derived
CD4+CD90.1-
(Thy1b+) cells. Anti-IP-10-treated mice had a
significant reduction of both encephalitogenic
Thy1a+ and Thy1b+
host-derived CD4+ cells accumulating in the CNS
(Table II). Differential CD45 staining intensity coupled with F4/80
staining has been used to distinguish between infiltrating macrophages
and resident microglia. Infiltrating macrophages are
CD45highF4/80+, while
resident microglial cells are
CD45lowF4/80+
(34). Our results indicate that most of the total CNS
mononuclear cells from the spinal cords of anti-IP-10-treated mice
were resident microglial cells, not infiltrating macrophages. In
contrast, 20% of the total CNS mononuclear cells from the control
mice were CD45bright, corresponding to
infiltrating macrophages. The relative increase in resident microglial
cells for the anti-IP-10-treated group does not indicate an
increase in microglial cell infiltration, rather it is indicative of an
increase in relative percentage due to the lack of other infiltrating
cell types.
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We asked whether disease protection in anti-IP-10-treated mice
could result from a defect in T cell activation and differentiation.
When control-treated mice showed peak clinical EAE, draining lymph node
cells and splenocytes from both control- and anti-IP-10-treated
mice were stimulated in vitro with PLP139–151 to
determine their ability to produce cytokines. The results in Fig. 6 indicate that there was no defect in
anti-IP-10-treated cells isolated from peripheral lymph nodes
(A and C) or spleen (B and
D) to produce IFN- (A and B), IL-2
(data not shown), or proliferate in response to specific Ag compared
with control-treated cells (C and D).
Collectively, these results indicate that IP-10 and its specific
receptor, CXCR3, are expressed in the CNS during development of acute
EAE and that inhibition of IP-10 with in vivo Ab therapy ameliorates
clinical disease by preventing mononuclear cell accumulation in
the CNS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokine-induced recruitment of leukocytes into the tissue is thought
to be an essential step in the pathogenesis of tissue-specific
autoimmune inflammatory disease progression (35). We
(17, 18) and others (14, 16, 36, 37) have
demonstrated a relationship between production of chemokines in the CNS
and development of acute and relapsing EAE. Here we report the
biological significance of IP-10 expression in the CNS and the
regulation of subsequent acute EAE. CD4+
encephalitogenic T cells can be detected in the CNS as early as 3–24 h
posttransfer (38, 39) (our unpublished observation) and
can stimulate resident cells, through TNF- and IFN-, to express
CCL5 (RANTES), MIP-1, MCP-1, and IP-10 (36, 37),
resulting in additional mononuclear cell recruitment. IP-10 expression
in the CNS of mice induced to develop EAE occurs well before the onset
of clinical symptoms (Fig. 2A) and has been localized to
astrocytes that form the perivascular-parenchymal boundary
(36).
The expression of CNS IP-10 mRNA (Fig. 2A) and protein (Fig. 1), along with the expression of CXCR3 mRNA by encephalitogenic Th1
cells isolated from the target organ during clinical disease (Fig. 2B), led us to hypothesize that IP-10 was responsible for
mononuclear cell accumulation resulting in subsequent disease
development. By administering neutralizing IP-10 antisera, we were able
to decrease clinical disease, lower disease incidence, and delay
clinical onset. Anti-IP-10 treatment decreased mononuclear cell
accumulation in the CNS (Fig. 5). We believe that because mononuclear
cells did not receive IP-10-mediated signals to accumulate in the CNS
there was an apparent increase in the total number of peripheral
mononuclear cells (Fig. 5). This peripheral increase is most likely a
result of the neutralization of a chemotactic gradient and the
inability of cells to accumulate in the CNS rather than expansion of
peripheral lymphocytes, as there is no evidence of an increase in
peripheral lymphocyte proliferation or production of proinflammatory
cytokines (Fig. 6). The mechanism of the disease and pathology
inhibition appears to be reduction in the accumulation of
PLP139–151-specific autoreactive Th1 cells in
the CNS (Table II). The prevailing model of CNS T cell entry suggests
that activated T cells gain entry to the CNS, but only the Ag-specific
T cells are retained (38). It is possible that initial CNS
Ag-specific T cell migration does not require chemokine expression by
the target organ. However, we hypothesize that IP-10 expression is
required for Ag-specific T cell and other mononuclear cell
accumulation. In the absence of disease-inducing T cell accumulation,
the inflammatory signals that induce the accumulation of other cell
types such as host-derived T cells and monocytes would not be present.
This would potentially account for the failure to detect other
CNS-infiltrating mononuclear cells in the anti-IP-10-treated mice
(Table II).
IP-10 chemokine expression has also been reported in cerebral spinal
fluid of MS patients during MS attacks (29). In addition
to chemokine expression, CXCR3, the receptor for IP-10, was found to be
expressed on infiltrating lymphocytic cells in demyelinating MS brain
lesions (29). Given the correlation of chemokine/chemokine
receptor expression in active MS lesions and EAE, the production of
IP-10 in the target organ early during disease initiation may be one of
the important events regulating early trafficking and/or accumulation
of autoreactive cells within the CNS. It was recently shown that
infusions of IP-10 antisense oligonucleotides reduced acute EAE
severity in Lewis rats, further demonstrating an important role for its
production (40). However, IP-10 was not expressed in the
CNS during clinical EAE in IFN--deficient BALB/c mice when disease
was induced with whole myelin-basic protein (41). This
result suggests IP-10 expression is not required for EAE induction in
BALB/c mice; however, the requirement for IP-10 in various mouse
strains may differ. A possible explanation may include the type of
disease manifestation in individual mouse strains. Disease in BALB/c
mice develops as widespread demyelination and disseminated leukocytic
infiltration of the spinal cord, which differs from the focal
perivascular infiltrates seen in SJL/J mice (42). The CNS
infiltrate in BALB/c mice is primarily composed of
Gr-1+ neutrophils, whereas SJL/J infiltrate is
comprised of mononuclear cells with little or no polymorphonuclear
infiltrate (4, 42). Therefore, production of IP-10 in the
CNS and its role during disease initiation may differ between mouse
strains. Alternatively, IP-10 may function as a chemokine that
participates in CNS T cell accumulation, but does not necessarily
function alone. The production of IP-10 in SJL EAE may be acting
coordinately with other chemokines, especially MIP-1 given its known
role in EAE pathogenesis (17).
Several other inflammatory models have demonstrated the important role
chemokines have for directed cell migration. Administration of RANTES
antisera to mouse hepatitis virus (MHV)-infected C57BL/6 mice resulted
in a significant reduction in macrophage infiltration and demyelination
during MHV infection (43). Polyclonal Ab directed against
RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis
model (44). Neutralization of CXCL1 (MIP-2) and MIP-1
was shown to attenuate neutrophil recruitment in the CNS during
experimental bacterial meningitis (45). IP-10 was
shown to be important for host antiviral responses following infection
of the CNS with MHV, where treatment of mice with anti-IP-10
antisera led to increased mortality, delayed viral clearance, and
decreased CD4+ and CD8+ T
lymphocyte infiltration to the CNS (46).
Our present results suggest that temporal IP-10 expression governs the accumulation of autoreactive inflammatory T cells into the target organ, resulting in tissue damage and clinical progression of EAE. These findings open the possibility of using CXCR3 antagonists for organ-specific autoimmune disease therapy. We are currently assessing the function of other CXCR3 ligands, such as Mig and I-TAC, for their role in EAE development to understand the universality of a CXCR3 antagonist approach to the treatment of autoimmune disease.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William J. Karpus, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, W127, Chicago, IL 60611. E-mail address: w-karpus{at}northwestern.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; MS, multiple sclerosis; MIP, macrophage-inflammatory protein; TCA-3, T cell activation gene-3; MCP, monocyte chemotactic protein; IP-10, IFN--inducible protein-10; I-TAC, IFN-inducible T cell -chemoattractant; Mig, monokine induced by IFN-; GRO, growth-related oncogene; VLA-4, very late Ag-4; IBS, isotonic-buffered saline; NRS, normal rabbit serum; MHV, mouse hepatitis virus.
Received for publication January 16, 2001. Accepted for publication April 11, 2001.
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). The data shown are representative of two independent
experiments.
