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T Cell Depletion on Chemokine and Chemokine Receptor Expression in the Central Nervous System1
*
Department of Pathology and Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10461; and
Scripps Research Institute, La Jolla, CA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cells significantly reduced clinical and pathological signs of disease,
which was associated with reduced expression of IL-1ß, IL-6, TNF-
,
and lymphotoxin at disease onset and a more persistent reduction in
IFN-. In this study, we analyzed the effect of T cell
depletion on chemokine and chemokine receptor expression. In the CNS of
control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory
protein (MIP)-1, MIP-1ß, MIP-2, inducible protein-10, and monocyte
chemoattractant protein-1 were detected at disease onset, increased as
disease progressed, and fell as clinical signs improved. In T
cell-depleted animals, all of the chemokine mRNAs were reduced at
disease onset; but at the height of disease, expression was variable
and showed no differences from control animals. mRNA levels then fell
in parallel with control EAE mice. ELISA data confirmed reduced
expression of MIP-1 and monocyte chemoattractant protein-1 at
disease onset in T cell-depleted mice, and total T cell numbers
were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were
observed, and these were elevated in EAE animals. mRNAs for CCR2 were
also detected in the CNS of affected mice. Depletion of T cells
reduced expression of CCR1 and CCR5 at disease onset only. We conclude
that T cells contribute to the development of EAE by promoting
an inflammatory environment that serves to accelerate the inflammatory
process in the CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies in the principal animal model of MS, experimental autoimmune
encephalomyelitis (EAE), have implicated a role for
CD4+ T cells expressing a Th1-type cytokine
profile in disease pathogenesis (reviewed in Refs. 4 and
5). The entry of these cells into the CNS is thought to be
dependent on the expression of an activated phenotype (6),
and in the mouse the surface expression of the adhesion molecule
4ß1 integrin (VLA-4)
and the induction of matrix metalloproteinase-2 have been strongly
implicated in trafficking of these cells across the blood-brain barrier
(7, 8, 9). After interaction with cognate Ag, these cells are
then thought to release proinflammatory cytokines and chemokines that
initiate an inflammatory cascade resulting in breakdown of the
blood-brain barrier and the influx of inflammatory cells into the CNS
compartment (reviewed in Refs. 5 and 10).
During the acute phase of the disease, lesions are composed of
blood-derived lymphocytes and monocytes that form the perivascular
cuffs, with demyelination occurring at these sites of inflammation.
Although Ag specificity can be shown to reside within the T cell
population that expresses the TCR-ß, we and others have shown that
T cells that express the TCR are present in the CNS lesions in
both EAE and MS (11, 12, 13, 14, 15, 16, 17, 18, 19). In EAE, these cells are usually
localized at the lesion edge, and their numbers can be shown to
fluctuate in association with disease activity (11).
Although the precise function of these cells has not been defined, they
are known to release cytokines such as IFN- and IL-4 and to possess
potent cytotoxic activity including cytotoxicity toward
oligodendrocytes, the myelin-forming cells in the CNS
(20, 21, 22). In previous studies, we showed that depletion of
these cells immediately before disease onset significantly decreased
disease severity and that this effect was associated with a significant
reduction in mRNA levels for the cytokines IL-1, TNF-, and IL-6 in
the CNS at disease onset and a more persistent reduction in the
expression of IFN- throughout the disease course (23).
The role of these proinflammatory cytokines as important regulatory
factors in the initiation and maintenance of EAE has been well
established.
Recently, an important role for chemokines in defining the inflammatory
infiltrate in EAE has also been proposed (reviewed in Ref.
10). Chemokines are small cytokines that are classified
into different subfamilies depending on the positioning of conserved
cysteine motifs and signal to cells through binding to seven
transmembrane spanning G protein-coupled receptors. They have been
shown to selectively direct the migration of specific populations of
leukocytes into tissues and may also reversibly activate leukocyte
integrins to modulate leukocyte-endothelial cell interactions. In the
Lewis rat, intrathecal administration of antisense oligonucleotides
against mRNA for the chemokine cytokine-responsive gene 2/IP-10, which
is a chemoattractant for activated T cells, reduced the severity of EAE
(24). In the mouse, administration of neutralizing Abs to
macrophage-inflammatory protein (MIP)-1, which also chemoattracts
activated T cells, was found to significantly reduce clinical
expression of disease during the acute clinical episode
(25), whereas Abs to monocyte chemoattractant protein
(MCP)-1, a chemoattractant for cells of the monocyte/macrophage series
as well as activated T cells, selectively reduced clinical disease only
during the relapsing phase of the disease (26). In
myelin-specific T cell lines, expression of the chemokine TCA-3 was
found to correlate with encephalitogenic potential (27).
These data, together with studies that have examined the temporal
expression of chemokines in the CNS of animals with EAE, support an
important role for these factors in disease pathogenesis
(28, 29, 30, 31, 32, 33, 34).
In this study, we have examined the effect of T cell depletion
on chemokine expression in the CNS of animals sensitized to develop EAE
by the passive transfer of T cells reactive to myelin basic protein
(MBP). The results support previous findings that there is an
up-regulation in the CNS of most of the C-C chemokines during the acute
phase of the disease, and we further suggest that T cells
contribute to the development of EAE by functioning to promote a
proinflammatory environment in the CNS at the initial phases of lesion
formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice, 6–8 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed and maintained in a federally approved animal facility, and the Animal Care and Use Committee of Albert Einstein College of Medicine approved all protocols.
Immunization and induction of EAE
Mice were immunized with myelin basic protein (MBP) (Sigma, St. Louis, MO) as previously described (23). Draining lymph nodes from axial, brachial, and inguinal regions were removed after 10 days, and single-cell suspensions were made in complete medium consisting of RPMI 1640, 10% FCS, 100 µg/ml penicillin/streptomycin, 2 mM glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 0.1 M HEPES, and 1% nonessential amino acids (Life Technologies, Grand Island, NY). Cells at a concentration of 4 x 106 cells/ml were activated in vitro with 50 µg/ml MBP at 37°C in 8% CO2 for 4 days. For adoptive transfer, 5 x 107 cells in 0.1–0.2 ml were injected into naive syngeneic recipients via the lateral tail vein. Clinical expression of disease was graded on a clinical index (CI) scale of 0–5 as follows: grade 1, limp tail; grade 2, hind limb weakness; grade 3, plegia of both hind limbs; 4, plegia of three or four limbs; grade 5, moribund. Recipient mice first showed signs of EAE 6–8 days posttransfer. Animals in recovery were defined as a clear clinical improvement of at least one grade persisting for 24–48 h after a paralytic incident, and relapses were defined as a clear clinical worsening of at least one grade persisting for 24–48 h after a clinical remission. Animals were studied from days 0–20 posttransfer.
In vivo depletion of T cells
Culture supernatant of the hamster IgG mAb against pan TCR
(GL3) (gift of Dr. L. Lefrancois, University of Connecticut,
Farmington, CT) was collected, and the IgG was affinity purified using
an Affi-Gel protein A MAPS II kit (Bio-Rad, Hercules, CA). Mice were
injected i.p. on 2 consecutive days with 500 µg purified GL3 Ab given
in two equal doses, twice a day. Control groups of mice were
injected with an equivalent amount and volume of normal hamster Ig
(NHIgG, Accurate Chemicals, Westbury, NY).
Immunohistochemistry
For immunohistochemical analysis of the tissue, mice were
anesthetized by ether inhalation and perfused through the left
ventricle with 30 ml cold PBS and snap frozen in optimal cooling
compound. Sections (10 µm) were prepared from the snap-frozen tissue.
Sections were air-dried for 30 min and fixed in ice-cold acetone for 10
min at -20°C. Slides were rinsed in PBS followed by incubation in
0.3% H2O2 in methanol for
15 min. After three washings with PBS, the sections were blocked with
10% normal goat serum for 1 h. Slides were then incubated with
the following Abs for common leukocyte Ag CD45 (Boehringer Mannheim,
Indianapolis, IN), mAb for human MCP-1 (mIgG1), and polyclonal Abs for
mouse MIP-1ß (gift from Dr. Barbara Sherry, Picower Institute, NY)
overnight at 4°C, washed three times in PBS, and incubated with
species-specific biotinylated secondary Abs (Vector Laboratories,
Burlingame, CA) for 2 h at room temperature. Control tissues were
incubated with purified normal rat IgG (for CD45), purified normal
hamster IgG (for ), mouse myeloma protein MOPC 21(Sigma) (for
MCP-1), and prebleed rabbit serum (for MIP-1ß) and processed as
above. Slides for MCP-1 were washed three times in PBS and incubated
with an avidin-biotin-HPO complex (Vector Laboratories) diluted 1:100,
mixed, and incubated for 1 h at room temperature.
3,3'-Diaminobenzidine tetrahydrochloride (Vector Laboratories) was used
for chromogenic detection of peroxidase staining. After the final wash
with PBS, sections were dehydrated and mounted in Permount. For CD45
and MIP-1ß, slides were washed in PBS and incubated with secondary
reagents coupled to FITC or tetramethylrhodamine isothiocyanate
(Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 for
30 min, washed with water, and mounted in Aqua mount (Kirkegaard &
Perry Laboratories, Gaithersburg, MD).
Isolation of leukocyte infiltrates in the CNS
Sensitized mice that had been treated with either NHIgG or GL3 as above were perfused transcardially with 30 ml ice-cold PBS at various time points. The spinal cord was dissected free from the vertebral column and dissociated by passing through a stainless steel mesh grid. Leukocytes were isolated by Percoll density gradient centrifugation and a total count obtained with a hemocytometer.
Preparation of total RNA
After administration of a lethal dose of sodium pentobarbital, mice were perfused transcardially with 30 ml ice-cold PBS. The spinal cord was removed, and total RNA extracted using TRIREAGENT (Molecular Research Center, Cincinnati, OH).
Multiprobe RNase protection assay (RPA)
RPA using either the mCK-5 multiprobe template set for
chemokines or the mCR5 template set for chemokine receptors
(PharMingen) was performed according to the manufacturer’s
instructions (Ambion, Austin, TX). Briefly for chemokines,
[-32P]UTP-labeled antisense RNA transcripts
encoding lymphotactin, RANTES, eotaxin, MIP-1ß, MIP-1, MIP-2,
IP-10, MCP-1, and TCA-3, as well as two housekeeping gene products
GAPDH and large ribosomal subunit protein 32 (L32), were generated
using T7 RNA polymerase. An additional chemokine probe set was prepared
and used as described previously (35). For chemokine
receptors, antisense RNA transcripts encoding for CCR1, CCR1b, CCR2,
CCR3, CCR4, CCR5, and the two housekeeping genes L32 and GAPDH were
generated with the T7 RNA polymerase. From 10 to 20 µg RNA from each
sample were allowed to hybridize to the labeled probe for 20 h at
45°C. Single-stranded RNA was digested with an RNase A/T1 mixture
(Ambion), and the hybrids were analyzed on denaturing
urea/polyacrylamide gels. Protected fragments were visualized by
autoradiography and quantified by phosphorimaging the gels with the use
of a Storm 860 scanner and the Image QuaNT V3.01 software (Molecular
Dynamics, Sunnyvale, CA). For each sample, a ratio of the intensity of
the chemokine or chemokine receptor band was obtained using the value
of the band for ml32.
ELISA
Sandwich ELISAs for MCP-1 and MIP-1 were performed using kits
purchased from R&D Systems (Minneapolis, MN). Microtiter plates were
coated with affinity purified Abs according to the manufacturer’s
instructions. Mice were perfused as before, and the spinal cords and
brains were homogenized with 1 ml PBS containing 0.1 M PMSF. A standard
curve was established using recombinant chemokines and 50 µl of these
standard samples and samples of a positive control (supplied by the
manufacturer) and homogenates of spinal cord and brain were added to
the wells and incubated at room temperature for 2h. After washing,
peroxidase-conjugated Abs specific for mouse MCP-1 or MIP-1 were
added and incubated for 2h at room temperature. Plates were washed,
reacted with substrate for 30 min, stop solution added and read at a
wavelength of 450 nm. The sample values were calculated from the
standard curve.
Statistical analysis
all data are expressed as the mean ± SD and differences between groups were determined using ANOVA and probability values of <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In animals sensitized to develop EAE by the adoptive transfer of
MBP-reactive T cells, clinical expression of disease commences, with
rare exception, 7 days after transfer. Clinical signs worsen rapidly to
peak 
10 days posttransfer. The animals then enter a recovery phase
and are relatively free of clinical signs 20 days after transfer. To
determine the effect of T cell depletion on chemokine gene
expression in the CNS of mice with EAE, mice were treated with the mAb
GL3 that recognizes the TCR on days 4 and 5 posttransfer, or
with an equivalent dose and volume of NHIgG as a control. We have shown
previously that treatment with GL3 leads to transient depletion of
T cells from the spleen and peripheral blood, whereas treatment
with NHIgG has no effect on T cell populations (13).
At various time points during the disease, the spinal cords were
removed from saline-perfused animals and chemokine gene expression
determined by RPA. Normal age-matched controls were included in each
experiment, and animals from three different experiments were studied.
The results of the RPA are shown in Fig. 1
and the quantitative analysis obtained
by phosphorimaging of the gels in Fig. 2.
Bands of interest were identified by linear transgression analysis of
the gels, using the undigested probe as defined markers. In the CNS
samples from normal mice, no signal was detected for any of the
chemokines examined. In control EAE animals treated with NHIgG, low
level expression of all of the C-C chemokines examined was noted in the
samples harvested on day 6. At this time point, none of the animals
demonstrated any clinical signs of disease. On day 7, all of the
animals displayed clinical signs consistent with the onset of EAE
(CI = 1.0 ± 0.7, n = 6), and in these
animals a striking increase (3-fold) for all of the chemokines
tested was noted over that found at day 6. The levels of mRNA for all
of the chemokines detected increased further in the samples taken on
day 10, when all of the animals were paralyzed (CI = 4.0 ±
0.4, n = 6), correlating with the height of the acute
clinical episode. In animals sacrificed on day 20, when they had
recovered from the acute clinical episode (CI = 1.3 ± 0.4,
n = 5), mRNAs for these chemokines were dramatically
reduced.
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and 2). The
lack of a significant difference for IP-10 likely reflects the fact
that higher levels for this cytokine were obtained in the control group
in experiment 1 than in experiment 2, because within each experiment
the values were statistically significantly different. On day 10, a
time point corresponding to the peak of the acute clinical episode in
the animals treated with NHIgG, animals treated with GL3 showed a lower
disease severity (CI = 1.6 ± 0.4, n = 6).
However, no differences were noted in chemokine mRNA levels between the
two groups. mRNA levels for these chemokines then fell dramatically on
day 20 when clinical signs of disease were reduced (CI = 1.1
± 0.5, n = 4).
The prominent expression of MCP-1 in these animals was unexpected in
light of data from other models of EAE in the SJL mouse in which it was
found that MCP-1 expression was low in the acute phase of disease but
became more prominent in the relapsing phase of the disease
(25). Therefore, we repeated this experiment using a
different set of animals and a different multiprobe RPA set
(35). As shown in Fig. 3, in
control EAE animals, low level expression of mRNA for all of the
chemokines tested was detected at disease onset, peaked with peak of
disease, and fell substantially as the animals recovered. In animals
treated with GL3, there was a striking reduction in chemokine mRNA
expression at disease onset. Increased expression of all of the
chemokines was noted at the height of the disease but remained at lower
levels than that found in the control EAE animals.
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), with statistically significant
differences between each of the time points for all of the chemokines,
with the exception of IP-10 in which, as noted above, differences were
noted in the values between the two experiments. The considerable rise
in chemokine mRNA expression noted between day 6 and day 7 indicated
that we had successfully bracketed the onset of inflammation in the CNS
and supported the conclusion that expression of these chemokines plays
a role in the influx of inflammatory cells into the CNS compartment.
However, no preferential pattern for any of these chemokines was noted
that correlated with the onset, height, or recovery phase of the
disease process. Depletion of T cells led to reduced expression
of mRNA for all of these chemokines at disease onset, but the extent to
which chemokine expression was altered at later stages of the disease
process was variable from one experiment to another. Nevertheless,
these data support a role for T cells in regulating the early
stages of the inflammatory process by modulating chemokine expression
in the CNS.
Chemokine protein expression in CNS of control and T
cell-depleted mice
To analyze whether the changes noted in mRNA levels for MCP-1 and
MIP-1 were correlated with evidence of chemokine protein present in
the CNS in control EAE and T cell-depleted mice, an additional
group of animals was sensitized and treated as before, and MCP-1 and
MIP-1 protein levels in CNS homogenates were determined by ELISA.
The results are shown in Table I.
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3-fold but were not significantly different
from the nonsensitized controls. Between day 6 and day 7, the values
increased 5-fold from the day 6 values (mean CI = 1.1 ±
0.5, n = 10, p < 0.008). On day 10
(mean CI = 3.4 ± 1.0, n = 5), however, the values
increased only slightly and were not significantly different from the
day 7 time point. This was followed by a marked drop in levels on day
20 (mean CI = 1.1 ± 0.7, n = 11). Thus,
these levels were compatible with the mRNA data obtained by RPA, as
well as the clinical expression of disease. In the T
cell-depleted animals, no change from the nonsensitized control values
were found on day 6 (mean CI = 0, n = 6). On day 7
(mean CI = 0, n = 9) a 3-fold increase over the
day 6 values was obtained but was significantly less than that found in
the NHIgG control group for the same time-point
(p < 0.008). On day 10 (mean CI =
1.6 ± 0.4, n = 5), the values increased 5 fold
but were not significantly different from the values obtained in the
control group. The values then fell as the animals went into the
recovery phase, and on day 20 (mean CI = 0.8 ± 0.3) were
similar to what was found in the NHIgG controls. For MIP-1, protein
levels in the control EAE animals were again well correlated with
disease expression and with the results of the RPA analysis.
Interestingly, in the GL3-treated group MIP-1 levels remained low
during disease onset, but at the height of the disease no difference
from the control group was noted, in agreement with the data for MCP-1.
These values then fell for both groups as the animals recovered from
the acute clinical episode. Thus, treatment with GL3 led to reduced levels of both of these C-C chemokines at disease onset, but at later stages of the disease no differences were noted between the groups, even though the animals treated with NHIgG had a greater mean CI, in agreement with the pattern of expression for the mRNA.
Immunohistochemistry for MCP-1 and MIP-1ß
To assess the distribution and extent of MCP-1 and MIP-1ß
immunoreactivity in CNS lesions of control EAE mice treated with NHIgG,
frozen sections of lumbar spinal cord were stained by
immunohistochemistry. Tissues sampled during the height of disease
(days 9 and 10) demonstrated MCP-1 immunoreactivity in most
infiltrating inflammatory cells and a few radial glial cells (Fig. 4). However, although T cells were
detected at these same sites, we have not detected MCP-1 expression in
T cells by intracellular staining (data not shown).
Immunohistochemical staining of serial sections for MIP-1ß revealed a
similar pattern of staining, with the majority of immunoreactivity
detected in infiltrating inflammatory cells (Fig. 5).
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T cell-depleted mice
To determine whether the depletion of T cells affected the
expression of chemokine receptors in EAE mice, we used the mCR5 RPA
multiprobe set. The same RNA samples assayed for chemokines shown in
Fig. 1 were used and were quantified as above (Figs. 6 and 7).
In the CNS of normal SJL/J mice, low level constitutive expression of
CCR1 and CCR5 was detected (Fig. 6). For CCR3, we have shown that in
the SJL mouse the band for this chemokine migrates aberrantly, due to a
polymorphism within the second membrane-spanning region
(36). Quantitation for this mRNA species was based on the
predicted protected fragment of 156 nt. In control EAE mice (Fig. 7),
an increase in the expression of all three chemokine receptors was
noted immediately before disease onset, and a signal for CCR2 became
evident. No band for CCR4 was identified in these animals. At the time
of disease onset (day 7), a 2- to 5-fold increase in expression of
these chemokine receptors was observed. At the height of disease (day
10), in control EAE animals there were no apparent differences in
expression of CCR2, CCR3, and CCR5 noted between day 7 and day 10, and
only the expression of CCR1 peaked with peak of disease in CNS. As the
animals recovered, the expression of all these receptors was
decreased.
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T cell-depleted animals, mRNA levels
for CCR2, CCR3, and CCR5 were higher than those detected in the control
EAE mice. Lower level expression of all of these receptors was noted,
however, on day 20 when clinical evidence of disease was reduced.
Total counts of infiltrating leukocytes in spinal cords of control
and T cell-depleted mice
To determine whether the changes noted in chemokine and chemokine
receptor expression reflected differences in the inflammatory
infiltrates in the CNS, the number of infiltrating leukocytes was
determined. The results are shown in Table II. In control EAE mice, the total number
of infiltrating cells rose dramatically at disease onset and
increased with increasing severity of the clinical signs. In T
cell-depleted mice sampled on day 6, the number of infiltrating cells
was less than that found in the NHIgG-treated animals, but these values
were not statistically different. On day 7, the number of infiltrating
leukocytes increased in the GL3-treated animals, but was significantly
lower than in the NHIgG-treated group (p <
0.04). However, at the height of disease, the total number of
leukocytes detected in CNS of T cell-depleted mice was not
significantly different from that found in the CNS of the NHIgG
animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell depletion on
chemokine and chemokine receptor expression in the CNS of SJL/J mice
sensitized to develop EAE by the adoptive transfer of MBP-specific T
cells. Our data indicate that in the CNS of control EAE mice treated
with NHIgG, mRNAs for a wide range of chemokines are expressed at
disease onset that include RANTES, eotaxin, MIP-1, MIP-1ß, MIP-2,
IP-10, and MCP-1. Although none of these chemokines was detected in the
CNS of age-matched normal mice, low but detectable levels of these
chemokines were noted in the CNS before disease onset, and a prominent
increase was observed through peak clinical expression of the disease.
T cell depletion before the onset of disease by the mAb GL3
reduced the levels of all chemokines at disease onset on day 7, but no
significant differences were noted between groups at later stages of
the disease, due to the variability between one experiment and another,
even though treatment with GL3 led to significant reduction in clinical
signs of disease in all of the groups of animals tested, consistent
with our previous report. The differences detected in the levels of
MCP-1 and MIP-1 at disease onset by RPA analysis were confirmed by
ELISA.
The fact that chemokines are up-regulated in the CNS of animals
sensitized to develop EAE has been well documented in several different
animal models, as well as in myelin-reactive T cells activated in vitro
(29, 30, 31, 32, 33, 34). Our studies are in substantial agreement with
these data and further support the observations of Godiska et al.
(31) that a broad range of chemokine mRNAs, including
MIP-1, MIP-1ß, RANTES, IP-10, MCP-1, and SDF-1, are expressed
before the onset of clinical disease and remain elevated throughout the
course of the acute clinical episode. Taken together, these data
strongly support the conclusion that chemokine gene and protein
expression in the CNS is tightly regulated and correlates with disease
expression. However, we were not able to demonstrate preferential
expression of any of these chemokines in association with a specific
stage of the disease process, nor did we detect a bias toward the
selective expression of specific chemokines. Analysis of the protein
levels for MIP-1 and MCP-1 confirmed the results of the RPA data. A
prominent role for MIP-1 in disease progression would be consistent
with the data of Karpus et al. (25), who found that
neutralizing Abs to this chemokine significantly protected animals
against the acute phase of the disease. MIP-1 levels were also well
correlated with evidence of T cell infiltration into the spinal cord.
However, the presence of a strong signal for MCP-1 was somewhat
unexpected in light of data from proteolipid protein-induced EAE where
MCP-1 expression and function has been more strongly linked to the
chronic-relapsing phase of the disease (Ref. 26 ; see also
Introduction). MCP-1 is a potent chemokine for cells of the
monocyte/macrophage series, acting through the chemokine receptor CCR2,
as well as for certain populations of activated T cells
(37). We were not able to satisfactorily perform total
counts of monocyte/macrophages in the populations of infiltrating cells
isolated by Percoll gradient density centrifugation, because cell
numbers obtained by this technique were at variance with previous
pathological analysis of the representation of these cells in the
lesion (23). We attribute this to the more adhesive
properties of monocytes for extracellular matrix components that are
deposited during the course of the inflammatory process.
An additional interesting difference in chemokine expression between this model of EAE induced in the SJL/J mice by the passive-transfer MBP-reactive T cells and EAE induced by the active sensitization of SWR/J mice with MBP was noted in the expression of C10. No signal for this chemokine was noted in the experiments reported here; however, in the SWR/J mice a prominent signal for C10 was detected throughout the disease course, similar to that found in mice that had been actively immunized with myelin oligodendrocyte protein (38). This chemokine has been shown to promote the recruitment of macrophages to the CNS, with mRNA and protein for this chemokine localized to macrophages/microglia and foamy macrophages within demyelinating lesions, as well as in perivascular infiltrates and meninges.
Although several studies have addressed chemokine expression in animals
with EAE, less is known about chemokine receptor expression. Our
findings of constitutive expression of CCR1, CCR3 and CCR5 in the
normal CNS is in agreement with documented studies that have detected
low level expression of these receptors in normal brain tissues, as
well as CXCR4 (39, 40), suggesting a role for chemokine
signaling in CNS function. Increased expression of these receptors has
been noted in a number of different pathological states, including HIV
encephalitis (40, 41, 42), Alzheimer’s disease
(43), MS (44), and EAE in the rat
(45). In our study, in MBP-induced EAE in the mouse,
animals treated with NHIgG, the C-C chemokine receptors CCR1 and CCR5
increased 3-fold at disease onset. These receptors are thought to be
specifically expressed by T cells polarized to a Th1 phenotype and to
function as receptors for MIP-1, MIP-1ß, and RANTES
(46, 47, 48). CCR2 was also elevated at disease onset. CCR2 is
the receptor for MCP-1 and has been shown to be a major regulator of
induced macrophage trafficking in vivo (49). In EAE in
rats, it has been suggested that increased expression of CCR2 is
derived from infiltrating macrophages (45). Its role in
the chemoattraction of cells that have been polarized toward either a
Th1 or Th2-type cytokine profile remains unclear. We observed a similar
pattern of expression for CCR3. This receptor has been reported to
selectively chemoattract Th2-specific T cells (46, 47, 48). In
T cell-depleted animals, a significant reduction in CCR1 and
CCR5 at disease onset was noted; this may be attributed to reduced
numbers of leukocytes that infiltrated into the CNS of these animals
(Table II). However, levels for CCR2, CCR3, and CCR5 were higher at the
height of disease in T cell-depleted mice than in control EAE
mice. In culture, it has been shown that chemokine receptor expression
is down-regulated after activation (50, 51, 52). These data
might suggest, therefore, that the infiltrating inflammatory cells in
the CNS in the T cell-depleted animals have not been fully
activated, an observation that could correlate with the lower levels of
IFN- in these tissues (23).
In mice depleted of T cells, the most striking differences in
chemokine and chemokine receptor expression were noted during the early
phases of the disease process. These results would be in agreement with
our previous data on cytokine expression, where we showed that only
IFN- was significantly reduced at all stages of the disease
process (23). A role for T cells in the regulation
of the inflammatory response has been documented in several different
model systems. In mice infected with Listeria monocytogenes,
T cells have been shown to participate in the establishment of
protective immunity by supporting priming of bacterial Ag-specific
CD8+ cytotoxic T cells (53) and to
play an important role in the transition from the innate to the
acquired immune response in these animals (54). T
cells in this model have also been shown to protect against liver
damage (55) and to be required for neutrophil accumulation
in the pleural cavity of mice after LPS challenge (56).
D’Souza et al. (57) also showed that T cells
regulated cellular trafficking of lymphocytes and monocytes into the
pleural cavity of Mycobacterium tuberculosis-infected mice,
possibly by secretion of specific chemokines. The role of T
cells as a source of chemokines has not been well established, but
intraepithelial T cells have been shown to express MIP-1,
MIP-1ß, RANTES, and lymphotactin but not MCP-1 (58). We
have obtained similar data in human T cell clones that express
the V2 TCR (59). However, in mice challenged with
L. monocytogenes, DiTirro et al. (60) observed
that infected T cell knockout mice showed reduced levels of
MCP-1 that correlated with a delay in monocyte accumulation in the
liver. Whether T cells are the source of this chemokine in these
lesions is not known at the present time. Nevertheless, these data
support the conclusion that T cells can function as a potent
source of both proinflammatory cytokines and chemokines that may
influence trafficking of inflammatory cells into the CNS
(23).
Ongoing studies using EAE induced with an encephalitogenic peptide of
myelin-oligodendrocyte glycoprotein in C57BL/6 mice in which the gene
for the -chain has been inactivated demonstrated a significant delay
in disease onset compared with wild-type mice, accompanied by a marked
reduction in CNS inflammation (A. J. Rajan et al., manuscript in
preparation). These data add further support to the conclusion that in
EAE activation of T cells serves to promote and accelerate the
early stages of the inflammatory process, consistent with a role that
has been proposed for these cells in several infectious diseases
(61, 62, 63). An interesting question that remains to be
addressed is the nature of the signal(s) that lead to the activation of
these cells in this autoimmune disease. Remarkably little is known with
certainty about the Ags recognized by cells, but a response to
activated ß T cells as well as to stress-related proteins has been
extensively documented (reviewed in Ref. 64). Taken
together, these observations suggest the hypothesis that a subset of
T cells act as regulatory cells which, after activation by
appropriate stimuli, accelerates the inflammatory response through
regulating the secretion of proinflammatory cytokines and chemokines.
In immune responses directed against pathogens, this accelerated
response could provide a significant protective advantage, whereas in
inflammatory autoimmune processes this could lead to accelerated
disease progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alice J. Rajan, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; CI, clinical index; MBP, myelin basic protein; NHIgG, normal hamster IgG; MIP, macrophage-inflammatory protein; MCP, monocyte chemoattractant protein; GL3, culture supernatant of the hamster IgG mAb against pan TCR ; IP-10, inducible protein-10; RPA, RNase protection assay; L32, large ribosomal subunit protein 32.
Received for publication June 2, 1999. Accepted for publication December 6, 1999.
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