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Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and the Th1 cytokines IL-2, IL-12, and
IFN-
was similar. Lymphocytes isolated from the CNS of SJL mice
produced large amounts of IL-10. CNS lymphocytes from both strains of
mice produced IFN- in response to stimulation with Sindbis virus,
but not in response to myelin basic protein. These data suggest that
IL-10-producing CD4+ T cells are differentially recruited
to or regulated within the CNS of SJL mice compared with BALB/c mice
infected with Sindbis virus, a characteristic that may be related to
low levels of IL-4, and is likely to be involved in susceptibility of
SJL mice to CNS inflammatory diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inflammation in the central nervous system (CNS) is rigorously controlled by several known mechanisms and probably additional mechanisms yet to be identified. The blood brain barrier is a physical barrier that prevents entry of most immune cells. Activated T cells can pass through the blood brain barrier, but quickly exit the CNS in the absence of Ag recognition (3, 4). T cell stimulation by Ag present in the CNS is further inhibited due to low levels of MHC molecules, costimulatory molecules, and adhesion molecules, although cellular damage and inflammatory mediators can increase expression (5, 6).
SV infection results in varying levels of CNS inflammation depending on the strain of mouse infected. Compared with BALB/c mice, SJL mice display more extensive inflammation and this inflammation may persist for several weeks to months (7). SJL mice are also susceptible to several inflammatory autoimmune diseases of the CNS such as experimental autoimmune encephalitis (EAE) (8, 9) and Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination (10), while BALB/c and many other strains of mice are relatively resistant to these diseases. Both of these murine demyelinating diseases serve as models for the human demyelinating disease multiple sclerosis, and more complete understanding of the inflammatory process may enhance our general understanding of autoimmune diseases of the CNS.
EAE is induced by immunization with whole spinal cord or components of
myelin such as myelin basic protein (MBP) or proteolipid protein
together with CFA (8, 11). Paralysis is accompanied by inflammation
consisting of T cells, macrophages and B cells. The disease is mediated
by CD4+ Th1-type T cells that secrete inflammatory
cytokines such as IL-2 and IFN- (12). The inflammation persists for
several days, then resolves.
TMEV infects neurons and oligodendroglia of the brain and spinal cord. Resistant strains of mice clear virus during the acute phase of the infection. In SJL mice, virus clearance is incomplete resulting in persistent infection of macrophages and glial cells. In these mice, TMEV-specific CD4+ Th1-type T cells mediate a chronic inflammatory process that results in demyelination and paralysis (13, 14).
The differences in the development and regulation of immune responses within the CNS of SJL mice compared with BALB/c mice that increase susceptibility to inflammatory diseases involve genes primarily outside the H-2 region and are only partially defined (10). To gain a better understanding of these differences, we have examined the CNS inflammatory response in SJL and BALB/c mice during acute encephalitis induced by infection with SV, a virus that is not associated with late neurologic disease. The immune response to SV results in clearance of virus in both strains of mice, but the inflammatory response to SV is more severe in SJL mice and SJL mice have lower levels of IL-4 mRNA, fewer NK cells, and more IL-10-producing CD4+ T lymphocytes in response to SV infection, suggesting altered immune regulation in the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL/J and BALB/cJ mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Four- to ten-week-old female mice were inoculated intracerebrally with 1000 plaque-forming units (PFU) of the AR339 strain of SV in 0.03 ml HBSS containing 1% FBS. Virus was grown and assayed using BHK-21 cells.
Histology and immunohistochemistry
At various times after infection, mice were anesthetized with methoxyflurane and perfused with PBS. Brains, spinal cords, and spleens were removed. All or part of each brain and spinal cord was either placed in 4% buffered paraformaldehyde for subsequent embedding in paraffin or rapidly frozen, together with the spleen, in OCT compound (Tissue-Tek; Miles, Elkhart, IN). Sections of paraffin-embedded tissue were stained with hematoxylin and eosin or with luxol fast blue, coded, and examined for inflammation and demyelination. Inflammation was scored on coded slides using a scale of 0–3 in which 0 indicates no detectable inflammation, 1 indicates one or two small inflammatory foci per section, 2 indicates moderate inflammatory foci in up to 50% of x10 fields, and 3 indicates moderate to large inflammatory foci in >50% of x10 fields. Scores were increased by up to 1 point with abundant parenchymal cellularity.
Cryopreserved sections were cut from OCT-embedded tissue and
used for immunohistochemistry. Sections were blocked with 0.5% dry
milk in PBS, and endogenous peroxidase activity was quenched with
either 1% H2O2 in absolute methanol or 0.3%
H2O2 in 0.1% sodium azide. Sections were
incubated overnight at 4°C, with primary Ab diluted in PBS containing
0.5% dry milk, washed, and incubated with biotinylated secondary Ab
(Vector Laboratories, Burlingame, CA) in PBS/0.5% dry milk. The signal
was amplified using the Vectastain Elite ABC kit (Vector) and detected
with 3,3'-diaminobenzidine (Sigma, St. Louis, MO). For detection of
CD8, the signal was further amplified using TSA-Indirect Tyramide
Signal Amplification (DuPont NEN, Boston, MA). The primary Abs used in
this study are listed in Table I
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Sections were examined under code for the level of inflammation, the
proportion of positively staining cells in inflammatory lesions, and
the relative number of positively staining cells in the parenchyma.
Immunohistochemical staining was scored on coded slides using a scale
of 0–3 in which 0 indicates no positive cells, 1 indicates 5–20%, 2
indicates 20–60%, and 3 indicates 60–100% positive inflammatory
cells. Scores were adjusted by up to 1 point when the numbers of
positively staining cells in the parenchyma were considerably higher or
lower than average.
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Brain lymphocytes were isolated as previously described (15). Briefly, SV-infected mice were anesthetized with methoxyflurane and perfused with PBS. Brains were removed, homogenized through a mesh screen, and collected in HBSS containing 0.05% collagenase D (Boehringer Mannheim, Indianapolis, IN) and 10 µg/ml DNase I (Sigma). The brain homogenate was mixed at room temperature for 20 min, then allowed to settle for 20 min. The supernatant fluid was collected and layered onto a mixture of 75% Ficoll-Paque (Pharmacia, Piscataway, NJ) and 25% RPMI 1640 with 10% FBS. After centrifugation at 500 x g for 30 min, the overlying media and interface of tissue debris were removed, and cells in the remaining gradient media were washed with PBS/2% FBS. The pelleted cells were resuspended in PBS/2% FBS for flow cytometry or RPMI 1640/10% FBS for culture.
For isolation of spleen lymphocytes, spleens were removed from perfused mice, homogenized through a mesh screen, and collected in PBS. The homogenate was layered onto Lympholyte M density separation media (Cedarlane, Westbury, NY). After centrifugation, cells were collected from the interface, washed, and resuspended as described for brain lymphocytes.
Flow cytometry
For flow cytometry, 3–5 x 105 isolated
lymphocytes in 100 µl PBS/2% FBS were incubated for 1 h at
4°C with FITC- or phycoerythrin (PE)-conjugated Abs as listed in
Table I
at 0.1 µg/ml. Conjugated isotype-matched control Abs at the
same concentration were used to determine background staining. Stained
cells were washed, resuspended in 0.5 ml PBS/2% FBS, and analyzed
using a FACScalibur flow cytometer (Becton Dickinson, Mountain View,
CA). Debris was excluded and intact lymphocytes were included in the
analysis using forward and side light scattering gates, and
5,000–10,000 gated events were collected for each sample. Percentages
of positively staining cells were calculated by establishing quadrants
on log-scale scatterplots of FITC vs PE fluorescence, which separated
background staining from positive staining.
Cytokine mRNA expression
Brains from perfused mice were removed, frozen on dry ice, and
subsequently homogenized in PBS. RNA was extracted from 1/4 of whole
brain homogenate using RNA STAT-60 (Tel-Test "B", Friendswood, TX).
cDNA was synthesized from 10% of total RNA as previously described
(16) using avian myeloblastosis virus reverse transcriptase (Boehringer
Mannheim). Then, 5% of the cDNA was used for each PCR reaction that
contained 200 µM of each dNTP, 1 µM of each specific primer, 2.5 U
Taq polymerase (Boehringer Mannheim), and buffer as supplied
by the manufacturer. PCR was performed using a 9600 thermal cycler
(Perkin-Elmer, Cetus, Norwalk, CT). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA detection was used to control for varying
amounts of input RNA. The optimal number of cycles needed for
amplification in the linear range was determined for each cytokine and
for GAPDH. Standards were included with each PCR reaction to ensure
that amplification was occurring in the linear range. Primers and
probes for GAPDH, IL-1ß, IL-2, IL-4, IL-6, IL-10, TNF, and IFN-
and primers for the chemokines macrophage inflammatory protein-1
(MIP-1), MIP-1ß, and IFN--inducible protein-10 (IP-10) have
been described previously (16, 17). Primers and probe for IL-12 were:
sense, 5'-TCTGCAGAGAAGGTCACACTG; anti-sense,
5'-GACTTCGGTAGATGTTTCCTC; probe, 5'-CCTGATGAAGAAGCTGGTGCTGTAGTTCTC.
For cytokines, PCR products were identified and quantitated relative to
GAPDH as previously described (16) except that probes were labeled with
digoxigenin and detected with alkaline phosphatase-labeled Ab to
digoxigenin and the chemiluminescent substrate disodium
3-(4-methoxyspirodecan)phenyl phosphate (Boehringer Mannheim). For
chemokines, PCR products were visualized on gels by staining with
ethidium bromide.
Functional assays using brain lymphocytes
For determination of IL-10 secretion, lymphocytes isolated from
brains or spleens of infected mice were added to 96-well plates at
5 x 105/ml in RPMI 1640 plus 10% FBS with or without
5 µg/ml ConA (Sigma) or 100 ng/ml phorbol myristic acid (Sigma) plus
2 µg/ml ionomycin (Sigma). After incubation for 3 days, supernatant
fluids were collected and the amount of IL-10 was determined by ELISA
using capture and detecting Abs shown in Table I following the protocol
provided by the manufacturer. rIL-10 (PharMingen, San Diego, CA) was
used to generate a standard curve.
For determination of Ag specificity, mononuclear cells were isolated
from spleens of uninfected mice and T cells were depleted by treatment
with rabbit anti-Thy1 antiserum (1:20; Cedarlane) at 4°C followed
by incubation at 37°C with Low-Tox-M rabbit complement (1:10;
Cedarlane). Lysed cells were removed using Lympholyte M as described
above. Lymphocytes isolated from brains of infected mice (5 x
105/ml) were combined with autologous T cell-depleted
spleen cells (2.5 x 106/ml) and incubated with or
without UV-inactivated SV equivalent to 2 x 107 or
8 x 107 PFU/ml or with human MBP at 25 µg/ml. After
incubation for 3 days, supernatant fluids were collected and IFN-
production was analyzed by ELISA (Endogen, Woburn, MA).
Detection of apoptosis
Brains and spinal cords removed from perfused mice were fixed in 4% paraformaldehyde and embedded in paraffin. Deparaffinized sections were subjected to terminal deoxynucleotidyltransferase-mediated UTP nicked end-labeling (TUNEL) as described previously (18). This technique detects endonucleolytically cleaved chromosomal DNA characteristically found in the nuclei of apoptotic cells. Staining was quantitated on coded slides by scoring the proportion of positive cells in inflammatory loci on a scale of 0–3 as for immunohistochemistry and the relative number of positive cells in the parenchyma on a scale of 0–3 in which 0 indicates no positive cells and 3 indicates the maximum number seen among all sections. These two scores were averaged for each section, then adjusted by up to 1 point if background staining was unusually high or low.
For annexin V staining, lymphocytes were isolated from brain and stained with PE-conjugated anti-CD3e for flow cytometry as described. Cells were then washed with PBS and incubated with FITC-conjugated annexin V diluted 1:10 in manufacturer-provided binding buffer from an Apoptosis Detection Kit (R & D Systems, Minneapolis, MN). Cells were diluted with binding buffer and analyzed immediately by flow cytometry as described.
Plaque assay
Brains were removed from perfused mice, frozen on dry ice, and stored at -80°C. Thawed brains were homogenized in cold PBS to make 10% (w/v) homogenates, and dilutions were prepared immediately in DMEM containing 1% FBS and plaqued on BHK-21 cells.
ELISA and plaque neutralization assay for serum Ab
Blood was collected from anesthetized mice, and serum was
isolated using microtainer serum separation tubes (Becton Dickinson).
For ELISA, enzyme immunoassay microtitration plates (Costar,
Cambridge, MA) were coated with polyethylene glycol-precipitated SV at
3 µg/ml. Serial dilutions of serum ranging from 1:10 to 1:3000 were
added to coated plates. Specific Ab was detected with horseradish
peroxidase-conjugated anti-mouse Ig, IgG1, or IgG2a (see Table I)
followed by O-phenylenediamine dihydrochloride (Sigma).
Relative titers were calculated from dilutions in the linear range
using a hyperimmune serum as a standard.
For neutralization assays, serial dilutions of serum were incubated with a known amount of SV for 30 min at 37°C. PFU/ml were measured on BHK-21 cells, and dilutions of serum needed for 50% plaque reduction were determined.
Statistical analysis
Student’s two-tailed test was used to assess significance of differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inflammation in the brains and spinal cords of SV-infected SJL and
BALB/c mice was assessed by routine histology and by
immunohistochemistry at various times after infection. Total
inflammation was quantitated using the number and size of inflammatory
lesions in each tissue section (Fig. 1A). Inflammation was greater
in SJL mice (day 5, p < 0.002; day 8,
p = 0.02) than in BALB/c mice. Inflammatory lesions
were first detected on day 3, and the level of inflammation reached a
peak on day 5 in both strains. However, at the time of maximal
inflammation, SJL mice had extensive infiltration of mononuclear cells
typically seen during fatal infection with a neurovirulent strain of SV
(Fig. 1B), while BALB/c mice showed only moderate
inflammation typical of nonfatal infection (Fig. 1C) (16).
In BALB/c mice, the inflammatory response was essentially resolved 2–3
wk postinfection (PI), but remained at moderate levels in SJL mice
until 4–5 wk PI in most mice and for several weeks longer in a few
mice. By 2 wk PI, inflammation in BALB/c mice consisted mostly of
increased cellularity in the parenchyma. In contrast, SJL mice
continued to have significant perivascular cuffing for the duration of
the inflammatory response. No demyelination was observed in either
strain at any time point.
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). Cells
expressing the B cell marker B220 were present at similar levels in SJL
mice and BALB/c mice, and the levels remained moderate through 4 wk PI.
Macrophages, identified by reactivity with the Ab F4/80, increased in
number during the first week PI, and levels were similar in SJL and
BALB/c mice. Cells expressing CD8 were also present at similar levels
in both strains. In contrast, cells expressing CD4 were more abundant
in SJL mice than in BALB/c mice. Many inflammatory lesions in brain and
spinal cord sections from SJL mice consisted of 60–80%
CD4+ T cells (Fig. 3A), while CD4+ T
cells made up only 20–30% of cells in lesions from BALB/c mice (Fig. 3B). The levels of CD4+ T cells in BALB/c mice
were similar to those seen in C57BL/6 mice and BALB/cBy mice (data not
shown). The representation of CD4+ T cells was notably
higher in SJL mice than in BALB/c mice at all time points with
detectable inflammation and remained at near maximal levels from 7 to
25 days PI (Fig. 2).
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Lymphocytes were isolated from the brains of SV-infected SJL and
BALB/c mice by density gradient separation and characterized by flow
cytometry. This allowed for a more quantitative characterization of the
types of immune cells present in the brains of these mice. Isolated
cells were double-stained for expression of CD3 together with CD4, CD8,
B220, ß or 
TCR, or a pan NK cell marker. The proportion of
isolated lymphocytes expressing each marker was determined at time
points between 4 and 14 days PI (Fig. 4).
Because debris from brain tissue remained in the cell preparations,
only cells with light scattering characteristics of lymphocytes were
analyzed. At most time points, 70–80% of the cells were identifiable.
However, on day 4 PI only 50% of the cells were identifiable due
either to the presence of a larger proportion of unidentified cells or
to the presence of proportionately more debris within the light
scattering gate because fewer lymphocytes were isolated on day 4 PI.
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A). By day 10, the proportion of
CD3+ cells was similar in both strains. Parallel to this
observation, the proportion of lymphocytes that were NK cells was
higher in BALB/c mice (day 6, p = 0.002) (Fig. 4A), consistent with earlier studies showing that SJL mice
have low, poorly inducible NK activity (19). In contrast to what was
seen with immunohistochemistry, the percentage of B220-expressing cells
was very low in both strains at all times examined (data not shown).
Staining for IgM resulted in similarly low levels of positive cells,
indicating that B cells may not be isolated efficiently by the
technique used.
The composition of the CD3+ cells was further studied by
staining for CD4 or CD8 (Fig. 4B). The proportion of
CD4+ and CD8+ cells was similar in the two
mouse strains through day 7 PI. After day 7, the proportion of
CD4+ cells increased in SJL mice, while the proportion of
CD8+ cells increased in BALB/c mice. A late increase in the
proportion of CD8+ T cells in the brains of BALB/c mice was
also seen in earlier flow cytometry, but not in immunocytochemical,
studies (15, 20). Staining of CD3+ cells for ß TCR and
TCR showed that in both strains of mice the majority of
CD3+ cells expressed ß TCR with <10% expressing
TCR (data not shown).
Cytokine mRNA expression by resident and inflammatory cells in the CNS
To characterize further the CD4+ T cells present at
increased levels in SJL mice and to determine whether early responses
of resident CNS to infection were different, cytokine mRNA expression
was analyzed by semiquantitative RT-PCR of RNA isolated from brains of
infected mice (Fig. 5). Early expression
of IL-1ß, IL-6, IL-12, and TNF mRNAs, proinflammatory cytokines
likely to be important for recruitment of inflammatory cells, was
identical. Expression of the mRNAs of three chemokines, believed to be
important in the recruitment of T cells and the development of EAE,
MIP-1, MIP-1ß, and IP-10 (21, 22), was measured, and no
differences were seen (data not shown). At days 5 and 8 PI, after
initiation of the inflammatory responses, expression of IL-10 was
higher in SJL mice (day 5, p = 0.002). Expression of
IL-4 was consistently higher in BALB/c mice (day 1, p =
0.001; day 3, p = 0.01). Expression of IL-12, IFN-,
and TNF mRNAs increased to similar levels in both strains.
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). Secretion of IL-10 by lymphocytes
from BALB/c brains was undetectable, while secretion by lymphocytes
from SJL brains was high and similar to secretion by lymphocytes
isolated from spleens of both strains of mice.
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Earlier studies had suggested that autoimmune T cells recognizing
MBP were activated in SJL mice during SV infection (7). To determine
whether the increased numbers of CD4+ T cells seen in the
CNS of SJL mice were virus-specific or recognized the autoantigen MBP,
we tested the ability of CNS lymphocytes to recognize SV proteins and
MBP (Fig. 7). Lymphocytes isolated from
the brains of mice 10 days after infection were cultured with
autologous T cell-depleted spleen cells in the presence or absence of
UV-inactivated SV or MBP to stimulate T cells through the MHC class II
pathway. Ag recognition was measured by secretion of IFN-. Both SJL
and BALB/c lymphocytes secreted IFN- in response to SV in a
dose-dependent manner, and neither responded to MBP. No IFN-
secretion was detected in the absence of Ag. SJL lymphocytes secreted
higher levels of IFN- in response to SV than BALB/c lymphocytes,
approximately proportionate to the higher levels of CD4+ T
cells in the SJL brain lymphocyte population.
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T cells and macrophages present in CNS inflammation during EAE
undergo apoptosis during the recovery phase of the disease (23, 24). To
determine whether the increased inflammation seen in SV-infected SJL
mice was due to reduced death of infiltrating inflammatory cells,
apoptotic cells were quantitated by two methods during the second week
PI. First, brain and spinal cord sections from SV-infected SJL and
BALB/c mice were stained using the TUNEL technique (Fig. 8A). In tissue sections from
both strains, a small proportion of cells in inflammatory lesions
stained positively. In addition, TUNEL-positive cells with lymphocyte
morphology were scattered throughout the parenchyma. TUNEL staining was
scored on coded sections by identifying the proportion of positive
cells in inflammatory foci together with the relative numbers of
positive cells in the parenchyma and was similar during this period of
time in the two mouse strains.
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B). Annexin V binds to phosphotidylserine expressed on the
cell surface during the early stages of apoptosis, and the level of
binding can be measured using flow cytometry (25). A similar small
proportion of CD3+ cells from the brains of both mouse
strains stained with annexin V. Virus replication and clearance
Differences in virus replication or clearance could change the
degree or persistence of the inflammatory response. To assess this
possibility, infectious virus in brain homogenates was measured by
plaque assay at various times after infection (Fig. 9A). The levels of infectious
virus and the time course of viral clearance were identical between the
two strains of mice.
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B). In both strains, Ab levels
increased during the first 1–2 wk PI and remained high thereafter.
There were no differences in Ab titers at any time point. The levels of
SV-specific IgG1 and IgG2a were also determined by ELISA, and in both
strains the majority of Ab was of the IgG2a isotype (data not shown).
Levels of neutralizing Ab in the serum were determined by plaque
neutralization, and again, no differences were seen (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to Ag
stimulation in vitro, but SJL CNS T cells also produced large amounts
of IL-10. These studies demonstrate that CNS immune responses in SJL
mice have inherent differences from those in BALB/c mice and provide
insight into the pathogenesis of the inflammatory diseases to which SJL
mice are susceptible. CD4+ T cells were much more prevalent and persistent in the brains and spinal cords of SJL mice in response to SV infection. That this is a characteristic of SJL mice is suggested by the fact that prolonged CNS inflammation, without evidence of persistent infection, has also been reported in SJL mice infected with Semliki Forest virus, an alphavirus related to SV (26). The phenotypes of the cells responding to Semliki Forest virus infection were not characterized, but studies of inflammatory cells in the brains of TMEV-infected SJL mice have also shown fewer CD8+ and more activated perivascular CD4+ T cells than in other strains of mice (14, 27). Furthermore, CNS mononuclear inflammatory cells in SJL mice infected with SV are more likely to show evidence of proliferation than in BALB/c mice (28). Together, these data suggest that alterations in recruitment of CD4+ T cells into the CNS and regulation of their numbers after entry may contribute significantly to the generation of inflammatory disease in the brain and spinal cord.
The initial phase of inflammation induced by SV infection followed a
similar time course. However, a considerable proportion of the
lymphocytes found in BALB/c brains early in the inflammatory response
were NK cells, while NK cells were nearly undetectable in SJL brains.
The response to TMEV in SJL mice is also deficient in NK cell activity,
and this is postulated to contribute to the defect in early virus
clearance (29). Although NK cells and NK activity are prevalent in the
CNS of BALB/c mice early after SV infection, there is no evidence that
they are necessary for, or contribute to, SV clearance from the CNS
(30, 31). NK cells secrete IFN- and TNF, cytokines that may play
a role in the initiation and control of T cell responses (32). However,
analysis of cytokine mRNAs in the brain early after infection revealed
no differences in IFN- or TNF. It is possible that the absence of
NK cells in SJL mice may be involved in shifting the immune response to
one dominated by CD4+ T cells.
We also found no differences in mRNA expression for IL-1ß or IL-6 or
the chemokines MIP-1, MIP-1ß, or IP-10. The only difference was
lower IL-4 mRNA levels in SJL mice. This difference has also been
observed in TMEV-infected mice (33) and may be due to the known SJL
deficiency in CD4+ T cells expressing NK1.1, a subset that
produces IL-4 efficiently early after TCR cross-linking (34, 35).
Numerous studies have demonstrated the ability of IL-4 to promote the
development of Th2 responses while inhibiting the development of Th1
responses (36). IL-4 knockout mice have an increased susceptibility to
EAE with more intense CNS inflammation, suggesting that IL-4 also
down-modulates inflammatory processes in the CNS (37). Therefore, low
levels of IL-4 in the CNS may play an important role in the
predisposition of SJL mice to increased and prolonged inflammation.
In EAE and TMEV-induced demyelination, disease-mediating T cells are of
the Th1 subtype, secreting inflammatory cytokines such as IL-2 and
IFN- (12, 13). A role for antiviral CD4+ Th1 responses
has also been suggested by an increased proportion of TMEV-specific Abs
of the IgG2a rather than the IgG1 subclass (38). We measured cytokine
expression to determine whether the CD4+ T cells present in
the CNS of SJL mice during SV infection were also of the Th1 subtype. A
comparison of the overall levels of CNS cytokine mRNA expression during
the peak of T cell infiltration showed that expression of IL-2 and
IFN- mRNAs, cytokines typically expressed by Th1 cells, was not
elevated in SJL mice. However, expression of Th2 cytokines IL-4 and
IL-10 was different. IL-10 mRNA and protein were increased while IL-4
mRNA was decreased. The study of CNS cytokine mRNAs in response to TMEV
infection has also shown lower levels of IL-4 mRNA and higher levels of
IL-10 mRNA during TMEV-induced disease in SJL mice compared with BALB/c
mice (33).
The increased expression of IL-10 mRNA in SV-induced encephalitis was
due to T cells because large amounts of IL-10 were produced in culture
by lymphocytes isolated from brains of infected SJL, but not BALB/c,
mice. Isolated lymphocytes also secrete IFN- in response to
stimulation with SV in culture, and both strains of mice produced
antiviral Ab primarily of the IgG2a subclass. Thus, the
CD4+ T cells present at increased levels in the CNS of SJL
mice during SV infection do not appear to be predominantly Th1 or Th2
cells. Although we do not know whether IFN- and IL-10 are produced
by the same population of T cells in SJL mice, CD4+ T cells
secreting this combination of cytokines have been reported by murine
clones suppressing autoimmune diabetes (39) and by human lines and
clones responding to chronic infection or primed by IL-12 (40, 41, 42).
The effect of IL-10 on inflammatory processes is complex. Although
generally regarded as a Th2 cytokine that down-regulates Ag
presentation and production of proinflammatory cytokines by macrophages
(43), IL-10 also up-regulates macrophage production of nitric oxide and
expression of FcR (44, 45). IL-10 inhibits the ability of microglial
cells to express MHC class II, to produce IL-12, to present Ag to T
cells and to activate astrocytes (46, 47, 48). IL-10 down-regulates T cell
responses in vivo. When given systemically IL-10 synergizes with IL-4
to inhibit delayed-type hypersensitivity responses (49, 50). IL-10 also
stimulates B cell proliferation and induces Ig secretion (51). Because
the presence of Ab is critical for control of SV replication (1),
inducing Ab production through IL-10 secretion may be a major role for
CD4+ T cells in the CNS of infected mice.
During monophasic inflammatory processes, IL-10 tends to be expressed at the highest levels late in inflammation. An increase in IL-10 expression in the CNS is observed during and after recovery from EAE (52). Apoptosis of activated cells is believed to be essential for down-regulation of most immune responses and appears to play an important role in regulation of inflammation in immune privileged sites (53). Apoptosis of T cells in inflammatory lesions of EAE along with increased production of IL-10 has been associated with decreasing inflammation and recovery from disease (23, 24, 52). IL-10 promotes activation-induced death of T cells and can prevent or treat autoimmune diseases, including EAE (54, 55, 56). Expression of IL-10 by T cells in SJL brains may, therefore, be a response to the high levels of inflammation and represent an attempt to down-regulate that process.
We found no evidence that SJL lymphocytes in the CNS exhibited increased apoptosis during the recovery phase, suggesting that the increased IL-10 produced did not effectively control the inflammatory process. Thus, the enhanced inflammation seen in SJL mice is most likely to result from increased entry of activated SV-specific IL-10-producing CD4+ T cells into the CNS of SJL mice from the periphery and perhaps local expansion of this population after entry. This increase in CNS inflammation is correlated with low levels of IL-4.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Diane E. Griffin, Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. E-mail address:
3 Abbreviations used in this paper: SV, Sindbis virus; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; GAPDH, glyceraldehyde phosphate dehydrogenase; IP, IFN--inducible protein; MBP, myelin basic protein; MIP, macrophage inflammatory protein; PE, phycoerythrin; PFU, plaque-forming units; PI, postinfection; TMEV, Theiler’s murine encephalomyelitis virus; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP nicked end labeling.
Received for publication June 17, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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