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,
*
Program in Neuroscience, Departments of
Pediatrics and
Microbiology, and
§
University of Iowa College of Medicine, University of Iowa, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice infected with the JHM strain of mouse hepatitis virus (MHV-JHM)3 develop acute and chronic demyelinating diseases (4, 5, 6). Although initial observations suggested that demyelination in this experimental infection was virus induced (7), recent experiments using irradiated mice or congenitally immunodeficient mice (mice with SCID) or with genetic disruption of the recombination-activating gene (RAG1-/-) clearly demonstrated the requirement for host immune function in this process (1, 4, 8). Normal T and B cells do not develop in SCID and RAG1-/- mice, and inoculation with the attenuated J2.2 strain of MHV-JHM (MHV-J2.2-v1) resulted in widespread infection of the white and gray matter without evidence of demyelination. Adoptive transfer of spleen cells from mice immunized with MHV into infected RAG1-/- mice resulted in extensive demyelination at 6–7 days posttransfer (p.t.), accompanied by a large influx of activated macrophages/microglia (1).
The cellular immune response to MHV-JHM infection in C57BL/6 (B6) mice has been extensively characterized. Both CD4 and CD8 T cells are necessary for virus clearance from the CNS of MHV-infected mice (9). Two CD8 T cell epitopes have been identified and are located within the surface (S) glycoprotein of MHV at residues 510–518 (S-510–518) and 598–605 (S-598–605), the former being the immunodominant of the two (10, 11). CD4 T cell epitopes are located in the transmembrane (M) protein of MHV (M-133–147) as well as the S protein (S-333–347, S-358–372) (12).
Although the role of MHV-specific T cells has not been directly addressed, data accumulated from several studies suggest that neither CD4 nor CD8 T lymphocytes are required for demyelination. Infection of mice genetically deficient in either MHC class I (ß2-microglobulin-/-) or MHC class II (Aß-/-) expression with MHV-J2.2-v1 resulted in diminished virus clearance in all mice, with demyelination detected in only some (13). These mice are deficient in CD8 or CD4 T cells, respectively. While these results suggest that neither CD4 nor CD8 T cells are indispensable for demyelination, the variation in the amount of demyelination observed in individual animals may reflect the ability of the immune system to compensate partially for a genetic defect during development. Depletion of either CD4 or CD8 T cells several days after infection with MHV did not result in a diminution in the amount of demyelination (14). However, the conclusion that neither CD4 nor CD8 T cells are required for demyelination was recently questioned by Lane et al. (15), who showed that less demyelination developed in mice in which the CD4 gene was genetically disrupted (CD4-/-).
In this study, as an alternative approach to dissecting the
requirements for different arms of the immune system in demyelination,
splenocytes depleted of either CD4 or CD8 T cells were adoptively
transferred into RAG1-/- mice infected with
MHV-J2.2-v1. Although depletion experiments have been used widely to
assess the role of different lymphocyte subsets in disease, their
interpretation is generally tempered by the possibility that a small
number of Ag-specific cells might remain in the depleted cell
population and contribute to the pathogenic process. Recent advances in
the ability to precisely measure the number of Ag-specific CD4 and CD8
T cells have made it possible to address this issue directly.
Previously, we have quantified the number of MHV-specific T cells
localized in the CNS by direct ex vivo assays using soluble MHC class
I/peptide tetramers, IFN-
enzyme-linked immunospot assays, and
intracellular IFN- assays. These methods have now been used to
quantify the number of MHV-specific CD4 and CD8 T cells in the initial
donor splenocyte population and in the CNS of adoptive transfer
recipients at the time of harvest. The adoptive transfer model, in
conjunction with these methods to track T cells, provides a unique
experimental system for determining the extent to which activated,
MHV-specific CD4 and CD8 T cells have nonredundant roles in
virus-induced demyelination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The neuroattenuated variant of MHV-JHM, strain J2.2-v1 (16), was generously provided by Dr. J. Fleming (University of Wisconsin, Madison, WI). Virus was grown and titered as previously described (17). Wild-type MHV-JHM was used to immunize wild-type B6 donor mice, as previously described (1).
Animals
Pathogen-free B6 mice were obtained from the National Cancer Institute (Bethesda, MD). RAG1-/- mice were obtained from The Jackson Laboratory (Bar Harbor, ME). No mature B or T lymphocytes are produced in RAG1-/- mice (18).
Adoptive transfer
RAG1-/- mice were infected with 1 x 103 PFU MHV-J2.2-v1 in 30 µl by intracranial injection (8). Adoptive transfer of splenocytes from B6 mice immunized i.p. with wild-type MHV-JHM to infected RAG1-/- mice was performed as previously described. Demyelination was maximal when cells were transferred to infected RAG1-/- mice 3 days p.i. No infectious virus could be detected by plaque assay in the transferred cells (1). A total of 74 MHV-J2.2-v1-infected RAG1-/- mice was used in these experiments. A total of 23 mice received undepleted donor cells, 19 mice received CD8 T cell-enriched donor cells, 24 mice received CD4 T cell-enriched donor cells, and 5 mice received donor cells depleted of both T cell subsets. Three RAG1-/- mice infected with MHV and harvested at day 10 p.i. without cell transfer served as controls. Clinical evaluation was based on the following scoring system: 0, asymptomatic; 1, limp tail; 2, wobbly gait with righting difficulty; 3, hind-limb weakness; 4, hind-limb paralysis; 5, moribund/dead.
Antibodies
mAbs F4/80 (macrophage-specific Ab, CI:A3-1; Serotec, Oxford,
U.K.) and 5B188.2 (Ab to MHV-JHM nucleocapsid protein; kindly provided
by Dr. M. Buchmeier, The Scripps Research Institute, La Jolla, CA) were
used for immunohistochemical labeling of macrophages/microglia and
virus Ag, respectively. Ab to the Fc receptor (2.4G2) and
FITC-conjugated Abs to CD4 (GK1.5) and CD8 (Lyt-2) Ags were obtained as
previously described (19). For depletion studies,
anti-CD4 Ab RL172 and anti-CD8 Ab 3.168 were kindly provided by
Dr. John Harty (University of Iowa, Iowa City, IA).
Complement depletion
Donor splenocytes depleted of CD4 or CD8 T lymphocytes were prepared for adoptive transfer by two rounds of complement lysis, as previously described (20). Greater than 99% depletion of CD4 or CD8 T cells was achieved, as assessed by FACS. No adjustment was made in the total number of donor cells delivered, since >90% of the initial cell population was recovered after depletion.
Histology
After perfusion of mice with PBS, brains and spinal cords were bisected in the midsagittal plane. Before embedding in paraffin, half of each sample was fixed in 10% normal buffered Formalin and half in Histochoice fixative (Amresco, Solon, OH). For examination of myelin and cell morphology, 8-µm sections were stained with luxol fast blue (LFB) and counterstained with hematoxylin and eosin.
Immunohistochemistry
Following hydration, 8-µm sections were permeabilized with 0.1% Triton X-100, blocked with CAS Block (Zymed Laboratories, San Francisco, CA), and incubated with primary Ab (F4/80 diluted in 1% normal goat serum at 1/200, 5B188.2 at 1/2000) overnight at 4°C. After washing, sections were incubated with biotinylated goat anti-rat (F4/80) or goat anti-mouse (5B188.2) (Jackson ImmunoResearch, West Grove, PA) diluted at 1/100 for 1 h. at room temperature. Following rinsing, avidin-HRP was applied using the ABC kit (Vector, Burlingame, CA), as directed by the manufacturer. The final substrate utilized for the staining reaction was 3,3'-diaminobenzidine (Sigma, St. Louis, MO).
Imaging
Images of stained spinal cord sections were digitalized using an Optiphot charge-coupled camera attached to a Leitz diaplan light microscope. Quantification of demyelination was done, as previously described (21), using Vtrace software (Image Analysis Facility, University of Iowa). Acquisition of all images was done at the University of Iowa Central Microscopy Research Facility.
FACS analysis
Lymphocytes were prepared from the CNS, as previously described
(22), and stimulated with peptide-coated CHB3 or EL-4
cells for Ag presentation to CD4 and CD8 T cells, respectively, in the
presence of monensin (Golgistop; PharMingen, San Diego, CA). Peptides
corresponding to the CD4 (M-133–147; I-Ab
restricted) and CD8 (S-510–518, H-2Db
restricted; S-598–605, H-2Kb restricted) T cell
epitopes were used at a final concentration of 5 and 1 µM,
respectively. After incubation for 4 h at 37°C, cells were
washed, blocked with 10% rat serum and anti-FcRII Ab 2.4G2
(1:200), and surface stained for CD4 and CD8 Ag (FITC-coupled mAb
GK1.5 or Ly-2, respectively). Cells were fixed and permeabilized using
the Cytofix/Cytoperm kit according to the manufacturer’s instructions
(PharMingen), stained for intracellular IFN- using PE-conjugated
anti-IFN- (PharMingen), and analyzed using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). The average number of
splenocytes isolated from donor spleens following i.p. immunization was
1.4 x 108 cells. Lymphocytes harvested from
the brains of two to three mice were pooled together before determining
the number of Ag-specific T cells. The range of lymphocytes harvested
from the pooled brain populations was 
1 x
106 to 2 x 106
(depending on time of harvest). The absolute number of Ag-specific
cells was calculated by multiplying the fraction of Ag-specific cells
by the fraction of CD4 or CD8 T lymphocytes by the total number of
cells per brain or spleen. Negative controls were splenocytes from
naive mice incubated with MHV-specific peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous experiments have shown that the MHV-specific CD8 T
cell response is directed at two epitopes (S-510–518 and S-598–605),
whereas the CD4 T cell response is dominated by cells recognizing
epitope M-133–147 (23). In the initial set of
experiments, the relative proportion and temporal appearance of
virus-specific CD4 and CD8 T cells in the CNS were quantified using
MHV-J2.2-v1-infected RAG1-/- mice following
adoptive transfer. Donor splenocytes were isolated from mice immunized
i.p. with live wild-type MHV-JHM at 6 days p.i. To identify the number
of Ag-specific CD4 and CD8 T cells within the donor population,
intracellular staining for IFN- was performed following in vitro
stimulation with peptides corresponding to known CD4 and CD8 T cell
epitopes, as described in Materials and Methods. Following
immunization, a low percentage of T cells responding to these three
epitopes was detectable in the initial spleen population (Fig. 1
A). No cells responding to
other CD4 T cell epitopes (S-333–347, S-358–372) could be detected in
this donor cell population (data not shown). From these data, we
calculated that there were 1–4 x 105
cells specific for each epitope per spleen. Since 5 x
106 splenocytes were delivered to each recipient,
7 x 103 donor cells responding to
M-133–147 and 1–2 x 104 CD8 T cells
specific for each CD8 T cell epitope were delivered to recipient mice
(Tables I and II).
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B,
Table II). Furthermore, 20% of CD4 T cells were specific for
the immunodominant CD4 T cell epitope, M-133–147 (Fig. 1B, Table I). Approximately
the same total number of Ag-specific CD4 or CD8 T cells was observed in
the infected CNS at this time p.t. as was detected in the transferred
donor population (Tables I and II). Of
note, the number of CD4 T cells responding to irrelevant peptide at
this time was elevated in both experiments. This response was not seen
in the donor populations or in cells harvested at day 7 p.t. from
the infected CNS. This result was not expected, since CD8 T cells, and
presumably CD4 T cells, rapidly turn off IFN- production in the
absence of stimulatory peptide (24). By 7 days p.t.,
a time when mice have developed demyelination, the frequency of epitope
M-133–147-specific CD4 T cells increased, while that of epitope
S-510–518-specific CD8 T cells decreased (Fig. 1C, Tables I and II). Approximately 20–25% of CD4 T cells were specific for the
immunodominant CD4 T cell epitope, while nearly 20% of CD8 T cells
were epitope S-510–518 specific. This shift in percentage of
Ag-specific CD4 and CD8 T lymphocytes from day 4.5 to day 7 p.t.
was not reflected in absolute numbers of cells, however, since on
average the absolute number of MHV-specific CD8 T cells in the CNS was
greater than that of CD4 T cells. Although the percentage of cells
responding to epitope S-598–605 remained low, the number of cells
responding to this epitope also increased from day 4.5 to day 7
p.t. These results show that a large increase in number of
virus-specific CD4 and CD8 T cells correlated with the development of
demyelination and clinical disease after adoptive transfer. Ag-specific T cells in CD4 and CD8 T cell-enriched adoptive transfer recipients
Next, the individual roles of MHV-specific CD4 and CD8 T cells in
demyelination were determined using the adoptive transfer model. For
this purpose, donor cells were depleted of either CD4 or CD8 T cells or
treated with complement only before adoptive transfer. Since depletion
of either subset resulted in only a small decrease in total number of
cells, no compensation was made in the number of cells transferred to
infected recipients. Greater than 99% depletion was observed for both
CD4 and CD8 T cells following complement lysis (data not shown). To
verify that the depleted T cell subset was not present in the CNS of
recipient mice, CNS lymphocytes were harvested and assayed for their
response to MHV T cell epitopes using IFN- intracellular staining.
At day 7 p.t., no CD8 T cells (total or MHV specific) were
isolated from the CNS of mice receiving CD4 T cell-enriched splenocytes
(Fig. 2). However, as expected, a
large percentage of CD4 T cells responded to epitope M-133–147 (Table III). Approximately the same absolute
number of MHV-specific CD4 T cells was present in the CNS of these mice
as was present in the CNS of recipients of undepleted populations
(compare Tables I and III).
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). In comparison with recipients of undepleted
splenocytes, the percentage and absolute number of S-510–518-specific
CD8 T cells in CD8 T cell-enriched recipients were lower. On day 7
p.t., only 11% of CD8 T cells isolated from the CNS of CD8 T
cell-enriched recipients responded to epitope S-510–518. Furthermore,
at times after day 11 p.t., the percentage of CD8 T cells
producing IFN- in response to S-510–518 remained low, at 10%.
These results are consistent with previous reports showing that CD4 T
cells are important for the trafficking, survival, or effector function
of CD8 T cells in the MHV-infected CNS (25). Clinical disease is different in recipients of CD4 and CD8 T cell-enriched donors
Although neurological disease developed in all mice following
adoptive transfer, a distinct difference in clinical signs between CD4
T cell- and CD8 T cell-enriched recipients was apparent. MHV-infected
RAG1-/- mice receiving CD8 T cell-enriched
donor populations did not exhibit as severe neurological symptoms at
day 7 p.t. as did mice receiving an equivalent number of
undepleted donor cells (Table IV). On
average, the appearance of hind-limb paralysis in CD8 T cell-enriched
recipients was delayed 4–5 days. Some animals survived until 15 days
p.t., with hind-limb paralysis as the predominant neurological sign. In
contrast, CD4 T cell-enriched recipients displayed severe neurological
dysfunction at 6 days p.t., often progressing to a moribund state soon
after clinical symptoms developed. Although there was a noticeable
variability in clinical disease between individual experiments, CD4 T
cell-enriched animals on average exhibited greater severity of symptoms
within each experiment (data not shown). Analysis of data from all
experiments showed that CD8 T cell-enriched and undepleted adoptive
transfer groups had significantly less clinical disease when compared
with the CD4 T cell-enriched group (Table IV).
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To determine whether levels of demyelination correlated with the
clinical differences observed between CD4 and CD8 T cell-enriched
recipients, spinal cord sections from each group were examined
histologically. LFB staining of spinal cord sections from recipients of
undepleted cells revealed extensive demyelination (Fig. 3A). Furthermore,
immunohistochemical labeling of F4/80 for macrophages/microglia showed
localization of these cells to areas of demyelination (Fig. 3B), as previously described (1).
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C). Quantification of lesion areas
throughout the spinal cords of these mice revealed significantly less
demyelination than detected in recipients of undepleted populations
(Table V). However, in marked contrast,
spinal cords from CD4 T cell-enriched recipients exhibited increased
cellularity when compared with those receiving undepleted or CD8 T
cell-enriched populations. Prominent areas of perivascular cuffing and
clusters of mononuclear cells were observed in both gray and white
matter (Fig. 3C). Many of these cells were
macrophages/microglia (Fig. 3D). Most notably, there was a
significantly greater infiltration of macrophages/microglia within the
gray matter than was observed in mice receiving undepleted populations
(compare Fig. 3, B and D).
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E), and quantification
demonstrated an equivalent percentage of demyelination when compared
with mice receiving undepleted cell populations (Table V). Only those
animals that exhibited neurological signs at day 7 p.t. were
analyzed for demyelination, to observe asymptomatic animals for longer
periods of time. Although infiltration of macrophages/microglia
into the white matter was detected, strikingly fewer
macrophages/microglia were seen in the gray matter than in that of CD4
T cell-enriched recipients (Fig. 3F). In animals that
survived more than 11 days p.t., the amount of demyelination increased,
with a concomitant increase in numbers of macrophages/microglia (data
not shown). These results show that clinical disease and demyelination
are not strictly correlated in the recipients of CD4 and CD8 T
cell-enriched splenocytes. Furthermore, the substantial increase in
demyelination observed between 7 days and later times p.t. in
recipients of CD8 T cell-enriched populations occurred in the absence
of any increase in the percentage of MHV-specific CD8 T cells in the
CNS (Tables III and V). CD4 T cell-enriched recipients are not deficient in viral clearance
Since both CD4 and CD8 T cells are necessary for effective
clearance of MHV from the CNS of infected mice (9), one
possible explanation for enhanced clinical disease in CD4 T
cell-enriched recipients is that virus is cleared less efficiently in
these mice. No difference in viral titer was observed in CD4 T
cell-enriched recipients relative to recipients of undepleted
splenocyte populations (Table V). Although CD8 T cell-enriched
recipients had greater amounts of virus recovered from the CNS at day
7 p.t., this difference was not present at later times, and was
not statistically significant. Furthermore, no difference in
localization or quantity of virus Ag was detected in CD4 or CD8 T
cell-enriched recipients in comparison with recipients of undepleted
spleen cells (data not shown). Therefore, differences in viral load or
localization do not explain the differences in clinical disease that
were observed.
MHV-induced demyelination is T cell dependent
To show directly that T cells were responsible for the induction
of demyelination in MHV-infected RAG1-/- mice
following adoptive transfer, the donor population was depleted of both
CD4 and CD8 T lymphocytes. Again, greater than 99% depletion was
achieved (data not shown). In these experiments, control mice received
undepleted spleen cells. Mice receiving cells depleted of both CD4 and
CD8 T cell lymphocytes died of encephalitis, similar to what was
observed in infected mice that did not receive transferred cells.
Histological examination showed scattered areas of demyelination in the
spinal cord (Fig. 4B).
Macrophages/microglia were detected throughout the spinal cord, but
most commonly had a spiny morphology resembling microglia, with minimal
numbers of rounded macrophages seen within the white matter (Fig. 4C). Virus Ag was abundant in the gray and white matter
(Fig. 4D), consistent with the encephalitis that developed
in these mice. Although the amount of demyelination detected was much
less than observed in recipients of undepleted or singly depleted T
cells (Table V), it was greater than the amount detected in
MHV-infected RAG1-/- mice in the absence of
transferred cells. Demyelination is detected in only a minority of
infected RAG1-/- mice not receiving transferred
cells and is always confined to the dorsal root-entry zones (Fig. 4A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results, showing that neither CD4 nor CD8 T cells are required
for demyelination, are in general agreement with a previous study using
mice congenitally deficient in CD4 or CD8 T cell function
(13). In contrast, Lane et al. (15) recently
showed that CD4 T cells are critical for neurological impairment,
inflammation, and demyelination in MHV-infected mice. Our results do
not necessarily disagree with the data in that report. The adoptive
transfer system inherently emphasizes the effector rather than the
initiation phase of the MHV-specific immune response. Both CD4 and CD8
T cells were activated before transfer since they were harvested from
B6 mice immunized to MHV 6 days previously. In contrast, much of the
diminished disease in CD4-/- mice reported by
Lane et al. was dependent on the delayed induction of inflammation by
CD4 T cells, particularly by the expression of the chemokine, RANTES.
In agreement with Lane et al., CD4 T cells very efficiently propagated
the pathological process in MHV-infected mice and, in the absence of
CD8 T cells, induced the infiltration of large numbers of macrophages
into both the gray and white matter (Fig. 3D). The extensive
infiltration of macrophages and microglia into the gray matter of these
mice may explain the propensity for more severe disease.
Our results show that 15–30% of CD4 and CD8 T cells in the CNS are MHV specific after adoptive transfer. Previous assessment of Ag specificity during MHV infection using soluble MHC class I/peptide tetramers revealed a large percentage of virus-specific CD8 T cells during acute and chronic disease, with one-third to one-half of these cells recognizing epitope S-510–518 (22, 26). The kinetics of appearance of MHV-specific CD8 T cells in the CNS are likely to be more rapid than in the natural infection in B6 mice, reflecting both the preexisting viral infection and the fact that these cells are already activated. Between 4.5 and 7 days p.t., during the development of neurological disease and demyelination, this number increases 5-fold, suggesting a rapid recruitment of Ag-specific cells to the infected CNS. MHV-specific CD4 T cells are also rapidly recruited to the CNS. Like MHV-specific CD8 T cells in the CNS of B6 mice with acute encephalitis, MHV-specific CD4 T cells also have an activated phenotype, enhancing their trafficking to the CNS (J. Haring, L. Pewe, and S. Perlman, unpublished data).
Following depletion of CD4 T cells from the donor cell population, the percentage of S-510–518-specific CD8 T cells within the infected CNS was decreased relative to recipients of undepleted cells analyzed in parallel. This reduction most likely reflects the requirement for CD4 T cell help for effective trafficking and/or survival of CD8 T cells within the CNS, as described previously (25). This diminished number of MHV-specific CD8 T cells did not affect the demyelinating process since a large increase in demyelination was seen at later time points in these mice. These results suggest that demyelination is induced when the number of MHV-specific CD8 T cells crosses a certain threshold, and that additional numbers of CD8 T cells do not enhance the process.
In another model of CNS demyelination, that induced by Theiler’s
murine encephalomyelitis virus (TMEV), depletion of CD4 or CD8 T cells
on a resistant background results in decreased virus clearance and
increased demyelination. More relevant to the present study are
analyses of the role of CD4 and CD8 T cells in mouse strains
susceptible to TMEV-induced demyelination (27). In these
strains, genetic deletion of CD4 T cells results in increases in
demyelination and neurological disease, whereas mice lacking CD8 T cell
function develop a significant amount of demyelination with minimal
clinical disease after infection with TMEV. Thus, in both MHV- and
TMEV-infected mice, CD4 and CD8 T cells are able to initiate
demyelination. Additionally, in both cases, the absence of CD8 T cells
results in less demyelination than observed in the absence of CD4 T
cells. In TMEV-infected mice, a relative paucity of symptoms is
observed in the absence of CD8 T cells, even in the presence of
significant demyelination. In marked contrast, depletion of CD8 T cells
results in an increase in clinical severity in MHV-infected mice (Table IV). The lack of clinical disease in TMEV-infected
CD8-/- mice has been attributed to maintenance
of axonal integrity (28). Axonal dysfunction is detected
in MHV-infected mice, but does not appear to explain the more severe
disease observed in recipients of CD4 T cell-enriched
populations.4
CD4 and CD8 T cells may contribute to demyelination by the same or
differing mechanisms. However, once the pathogenic process has begun,
the pathway to MHV-induced demyelination appears to be redundant, with
no single effector molecule being essential for demyelination. TNF-
,
IFN-, and NO are not required for MHV-induced demyelination
(29, 30, 31, 32). Furthermore, neither Fas/Fas ligand interactions
nor perforin-mediated cytolysis are necessary for this process
(33, 34). Consistent with these results, we have observed
no decrease in demyelination in infected
RAG1-/- mice after adoptive transfer of donor
cells from perforin-/- mice immunized with
MHV-JHM (data not shown).
Our results clearly show that T cells are most efficient at propagating the demyelinating process. However, cells that are not thymus derived may substitute in this process since nude rodents infected with MHV also develop demyelination (13). B cells are present in nude rodents, but in the present study, infusion of splenocytes depleted of T cells but not B cells resulted in only a small amount of demyelination. B cells appear to contribute to clinical disease in rats acutely and chronically infected with MHV (35, 36). Although B cells may not be important for initiation of demyelination, they may potentiate the process after initiation by Ag-specific CD4 or CD8 T cells.
A striking difference between recipients of CD4 T cell-enriched splenocytes and those receiving either undepleted or CD8 T cell-enriched populations is the increase in severity of clinical disease. Although the basis for this difference is not known with certainty, the number of macrophages in the gray matter of the CD4 T cell-enriched recipients is markedly increased compared with the other groups. It is clear from our studies and the reports of others that macrophages are key effector cells in the demyelinating process (3, 21, 37, 38). Our results show that either Ag-specific CD4 or CD8 T cells are sufficient for macrophage activation and subsequent demyelination to occur. Future studies directed at determining the precise mechanisms used by MHV-specific T cells to activate macrophages and those involved in destruction of myelin by these macrophages will be key to understanding virus-induced demyelination.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stanley Perlman, Department of Pediatrics, 2042 Medical Laboratories, University of Iowa, Iowa City, IA 52242.
3 Abbreviations used in this paper: MHV, mouse hepatitis virus; LFB, luxol fast blue; M, MHV-JHM transmembrane protein; p.i., postinoculation; p.t., posttransfer; RAG, recombination-activating gene; S, MHV-JHM surface glycoprotein; TMEV, Theiler’s murine encephalomyelitis virus.
4 G. Wu, A. Dandekar, L. Pewe, and S. Perlman. Axonal damage in mice with virus-induced demyelination. Submitted for publication.
Received for publication March 2, 2000. Accepted for publication May 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but requires TNF-. J. Immunol. 160:898. is required for viral clearance from central nervous system oligodendroglia. J. Immunol. 162:1641.-irradiated rats by transfer of naive lymphocyte subsets before infection. J. Neurovirol. 5:249.[Medline]
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 N. S. Butler, A. Theodossis, A. I. Webb, M. A. Dunstone, R. Nastovska, S. H. Ramarathinam, J. Rossjohn, A. W. Purcell, and S. Perlman Structural and Biological Basis of CTL Escape in Coronavirus-Infected Mice J. Immunol., March 15, 2008; 180(6): 3926 - 3937. [Abstract] [Full Text] [PDF]
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S. A. Stohlman, D. R. Hinton, B. Parra, R. Atkinson, and C. C. Bergmann CD4 T Cells Contribute to Virus Control and Pathology following Central Nervous System Infection with Neurotropic Mouse Hepatitis Virus J. Virol., March 1, 2008; 82(5): 2130 - 2139. [Abstract] [Full Text] [PDF]
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 R. Burrer, M. J. Buchmeier, T. Wolfe, J. P.C. Ting, R. Feuer, A. Iglesias, and M. G. von Herrath Exacerbated Pathology of Viral Encephalitis in Mice with Central Nervous System-Specific Autoantibodies Am. J. Pathol., February 1, 2007; 170(2): 557 - 566. [Abstract] [Full Text] [PDF]
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L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [Abstract] [Full Text] [PDF]
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D. Anghelina, L. Pewe, and S. Perlman Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis Am. J. Pathol., July 1, 2006; 169(1): 209 - 222. [Abstract] [Full Text] [PDF]
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A. M. Ercolini and S. D. Miller Mechanisms of Immunopathology in Murine Models of Central Nervous System Demyelinating Disease J. Immunol., March 15, 2006; 176(6): 3293 - 3298. [Abstract] [Full Text] [PDF]
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J. M. Gonzalez, C. C. Bergmann, C. Ramakrishna, D. R. Hinton, R. Atkinson, J. Hoskin, W. B. Macklin, and S. A. Stohlman Inhibition of Interferon-{gamma} Signaling in Oligodendroglia Delays Coronavirus Clearance without Altering Demyelination Am. J. Pathol., March 1, 2006; 168(3): 796 - 804. [Abstract] [Full Text] [PDF]
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Y. Liu, Y. Pu, and X. Zhang Role of the Mitochondrial Signaling Pathway in Murine Coronavirus-Induced Oligodendrocyte Apoptosis J. Virol., January 1, 2006; 80(1): 395 - 403. [Abstract] [Full Text] [PDF]
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L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman A Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Protein Enhances Virulence of an Attenuated Murine Coronavirus J. Virol., September 1, 2005; 79(17): 11335 - 11342. [Abstract] [Full Text] [PDF]
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A. A. Dandekar, K. O'Malley, and S. Perlman Important Roles for Gamma Interferon and NKG2D in {gamma}{delta} T-Cell-Induced Demyelination in T-Cell Receptor {beta}-Deficient Mice Infected with a Coronavirus J. Virol., August 1, 2005; 79(15): 9388 - 9396. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Viral Expression of CCL2 Is Sufficient To Induce Demyelination in RAG1-/- Mice Infected with a Neurotropic Coronavirus J. Virol., June 1, 2005; 79(11): 7113 - 7120. [Abstract] [Full Text] [PDF]
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K. C. MacNamara, M. M. Chua, P. T. Nelson, H. Shen, and S. R. Weiss Increased Epitope-Specific CD8+ T Cells Prevent Murine Coronavirus Spread to the Spinal Cord and Subsequent Demyelination J. Virol., March 15, 2005; 79(6): 3370 - 3381. [Abstract] [Full Text] [PDF]
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X. Zhong, H. Yang, Z.-F. Guo, W.-Y. F. Sin, W. Chen, J. Xu, L. Fu, J. Wu, C.-K. G. Mak, C.-S. S. Cheng, et al. B-Cell Responses in Patients Who Have Recovered from Severe Acute Respiratory Syndrome Target a Dominant Site in the S2 Domain of the Surface Spike Glycoprotein J. Virol., March 15, 2005; 79(6): 3401 - 3408. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Virus-Specific Antibody, in the Absence of T Cells, Mediates Demyelination in Mice Infected with a Neurotropic Coronavirus Am. J. Pathol., March 1, 2005; 166(3): 801 - 809. [Abstract] [Full Text] [PDF]
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Y. Liu and X. Zhang Expression of Cellular Oncogene Bcl-xL Prevents Coronavirus-Induced Cell Death and Converts Acute Infection to Persistent Infection in Progenitor Rat Oligodendrocytes J. Virol., January 1, 2005; 79(1): 47 - 56. [Abstract] [Full Text] [PDF]
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W. G. Glass, M. J. Hickey, J. L. Hardison, M. T. Liu, J. E. Manning, and T. E. Lane Antibody Targeting of the CC Chemokine Ligand 5 Results in Diminished Leukocyte Infiltration into the Central Nervous System and Reduced Neurologic Disease in a Viral Model of Multiple Sclerosis J. Immunol., April 1, 2004; 172(7): 4018 - 4025. [Abstract] [Full Text] [PDF]
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L. L. Pewe, J. M. Netland, S. B. Heard, and S. Perlman Very Diverse CD8 T Cell Clonotypic Responses after Virus Infections J. Immunol., March 1, 2004; 172(5): 3151 - 3156. [Abstract] [Full Text] [PDF]
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C. C. Bergmann, B. Parra, D. R. Hinton, C. Ramakrishna, K. C. Dowdell, and S. A. Stohlman Perforin and Gamma Interferon-Mediated Control of Coronavirus Central Nervous System Infection by CD8 T Cells in the Absence of CD4 T Cells J. Virol., February 15, 2004; 78(4): 1739 - 1750. [Abstract] [Full Text] [PDF]
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A. A. Dandekar, D. Anghelina, and S. Perlman Bystander CD8 T-Cell-Mediated Demyelination is Interferon-{gamma}-Dependent in a Coronavirus Model of Multiple Sclerosis Am. J. Pathol., February 1, 2004; 164(2): 363 - 369. [Abstract] [Full Text] [PDF]
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Y. Liu, Y. Cai, and X. Zhang Induction of Caspase-Dependent Apoptosis in Cultured Rat Oligodendrocytes by Murine Coronavirus Is Mediated during Cell Entry and Does Not Require Virus Replication J. Virol., November 15, 2003; 77(22): 11952 - 11963. [Abstract] [Full Text] [PDF]
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M. L. Moore, C. C. Brown, and K. R. Spindler T Cells Cause Acute Immunopathology and Are Required for Long-Term Survival in Mouse Adenovirus Type 1-Induced Encephalomyelitis J. Virol., September 15, 2003; 77(18): 10060 - 10070. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Protection Against CTL Escape and Clinical Disease in a Murine Model of Virus Persistence J. Immunol., August 15, 2003; 171(4): 2006 - 2013. [Abstract] [Full Text] [PDF]
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M. J. Trifilo, C. C. Bergmann, W. A. Kuziel, and T. E. Lane CC Chemokine Ligand 3 (CCL3) Regulates CD8+-T-Cell Effector Function and Migration following Viral Infection J. Virol., April 1, 2003; 77(7): 4004 - 4014. [Abstract] [Full Text] [PDF]
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C. C. Bergmann, B. Parra, D. R. Hinton, R. Chandran, M. Morrison, and S. A. Stohlman Perforin-Mediated Effector Function Within the Central Nervous System Requires IFN-{gamma}-Mediated MHC Up-Regulation J. Immunol., March 15, 2003; 170(6): 3204 - 3213. [Abstract] [Full Text] [PDF]
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W. G. Glass and T. E. Lane Functional Expression of Chemokine Receptor CCR5 on CD4+ T Cells during Virus-Induced Central Nervous System Disease J. Virol., December 6, 2002; 77(1): 191 - 198. [Abstract] [Full Text] [PDF]
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A. A. Dandekar and S. Perlman Virus-Induced Demyelination in Nude Mice Is Mediated by {gamma}{delta} T Cells Am. J. Pathol., October 1, 2002; 161(4): 1255 - 1263. [Abstract] [Full Text] [PDF]
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J. S. Haring, L. L. Pewe, and S. Perlman Bystander CD8 T Cell-Mediated Demyelination After Viral Infection of the Central Nervous System J. Immunol., August 1, 2002; 169(3): 1550 - 1555. [Abstract] [Full Text] [PDF]
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L. Pewe, J. Haring, and S. Perlman CD4 T-Cell-Mediated Demyelination Is Increased in the Absence of Gamma Interferon in Mice Infected with Mouse Hepatitis Virus J. Virol., June 14, 2002; 76(14): 7329 - 7333. [Abstract] [Full Text] [PDF]
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J. H. Dufour, M. Dziejman, M. T. Liu, J. H. Leung, T. E. Lane, and A. D. Luster IFN-{gamma}-Inducible Protein 10 (IP-10; CXCL10)-Deficient Mice Reveal a Role for IP-10 in Effector T Cell Generation and Trafficking J. Immunol., April 1, 2002; 168(7): 3195 - 3204. [Abstract] [Full Text] [PDF]
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L. Pewe and S. Perlman Cutting Edge: CD8 T Cell-Mediated Demyelination Is IFN-{gamma} Dependent in Mice Infected with a Neurotropic Coronavirus J. Immunol., February 15, 2002; 168(4): 1547 - 1551. [Abstract] [Full Text] [PDF]
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M. C. G. Marcondes, E. M. E. Burudi, S. Huitron-Resendiz, M. Sanchez-Alavez, D. Watry, M. Zandonatti, S. J. Henriksen, and H. S. Fox Highly Activated CD8+ T Cells in the Brain Correlate with Early Central Nervous System Dysfunction in Simian Immunodeficiency Virus Infection J. Immunol., November 1, 2001; 167(9): 5429 - 5438. [Abstract] [Full Text] [PDF]
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B. P. Chen, W. A. Kuziel, and T. E. Lane Lack of CCR2 Results in Increased Mortality and Impaired Leukocyte Activation and Trafficking Following Infection of the Central Nervous System with a Neurotropic Coronavirus J. Immunol., October 15, 2001; 167(8): 4585 - 4592. [Abstract] [Full Text] [PDF]
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M. T. Liu, H. S. Keirstead, and T. E. Lane Neutralization of the Chemokine CXCL10 Reduces Inflammatory Cell Invasion and Demyelination and Improves Neurological Function in a Viral Model of Multiple Sclerosis J. Immunol., October 1, 2001; 167(7): 4091 - 4097. [Abstract] [Full Text] [PDF]
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A. A. Dandekar, G. F. Wu, L. Pewe, and S. Perlman Axonal Damage Is T Cell Mediated and Occurs Concomitantly with Demyelination in Mice Infected with a Neurotropic Coronavirus J. Virol., July 1, 2001; 75(13): 6115 - 6120. [Abstract] [Full Text] [PDF]
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J. S. Haring, L. L. Pewe, and S. Perlman High-Magnitude, Virus-Specific CD4 T-Cell Response in the Central Nervous System of Coronavirus-Infected Mice J. Virol., March 15, 2001; 75(6): 3043 - 3047. [Abstract] [Full Text]
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