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Departments of
*
Neurology and
Immunology, Mayo Clinic/Foundation, Rochester, MN 55905; and
Celtrix Pharmaceuticals, Santa Clara, CA 95052
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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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More recent work has focused on the role of soluble mediators in the
development or prevention of TMEV-induced pathology. Treatment of SJL/J
mice with Abs to IFN-
resulted in an increased level of
demyelination (6). Likewise, infection of IFN-
receptor knockout mice on a background resistant to demyelinating
disease resulted in large demyelinated lesions and increased mortality
(7). Genetic mapping studies from the laboratory of Michel
Brahic have identified a region on chromosome 10 near the IFN- locus
that influences viral persistence (8). The type I IFNs,
IFN-
/ß, are also critical in the host’s defense against
Theiler’s virus. In the absence of the receptor to IFN-/ß, mice
do not live past the first few days of infection due to overwhelming
viral replication (7).
Administration of exogenous cytokines has also been shown to influence
demyelination. Experiments from our laboratory have demonstrated that
administration of IL-6 (9) and TNF- (10),
two cytokines that participate in inflammatory reactions, significantly
reduces demyelination in susceptible strains of mice. In contrast,
treatment of strains normally resistant to pathology with IL-1ß
peptide induced demyelination (11). Inoculation of
susceptible mice with IL-2-secreting tumor cells increased the
frequency of TMEV-specific pCTLs and prevented persistent infection
(12). These results, together with the data from the
knockout/depletion experiments, emphasize the complex nature of the
effect of cytokines on demyelination in this model.
TGF-ß1 and TGF-ß2 are potent immunoregulatory mediators whose
effects include inhibition of T and B cell proliferation, stimulation
of IL-1 and TNF- secretion by monocytes, and inhibition of class II
MHC expression (13). In vivo administration of TGF-ß1
reduces the occurrence of relapses in the experimental autoimmune
encephalomyelitis (EAE) model of multiple sclerosis
(14, 15, 16). In the present study, we examined the effect of
TGF-ß2 on the development of demyelination following intracerebral
infection with Theiler’s virus. SJL/J mice that received exogenous
TGF-ß2 had less demyelination and virus persistence compared with
infected control mice. Increased levels of virus-non-specific CTL
activity were observed in these mice compared with control mice. TUNEL
revealed that mice treated with TGF-ß2 had significantly fewer
apoptotic cells in the white matter compared with control-treated mice.
In addition, fewer macrophages were detected in the spinal cord white
of mice treated with TGF-ß2. The results are consistent with the
hypothesis that the mechanism of decreased demyelination may be by
reducing macrophage-mediated apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Daniel’s strain of TMEV was used in all experiments. The passage history of this virus has been described previously (17).
Animals
Female SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Handling of all animals conformed to the guidelines of both the National Institutes of Health and the Mayo Clinic/Foundation Animal Care and Use Committee.
Treatment of mice with TGF-ß2
Mice were i.p. injected with TGF-ß2 (gift from Celtrix Pharmaceuticals, Santa Clara, CA) at a dose of 0.1 µg beginning at either the day before infection with TMEV, or a dose of 1 µg beginning at day 15 postinfection (p.i.) with TMEV. This dose was used by Racke et al. and shown to be effective in the prevention and treatment of chronic EAE (16). Control mice received the vehicle (polyethylene glycol (PEG)) in lieu of TGF-ß2. Mice were treated three times per week for the duration of the experiment.
Infection, harvesting, and morphology of the CNS
At 4–6 wk of age, mice were intracerebrally infected with 2 x 105 PFU of TMEV in a total volume of 10 µl. Thirty-five days after infection, mice were perfused via intracardiac puncture with 50 ml of Trump’s fixative. Spinal cords were removed and processed for pathologic studies. Spinal cords were cut into 1-mm coronal blocks, and every third block was osmicated and embedded in glycol methacrylate, as previously described (18). Two-micron sections were prepared and stained with a modified erichrome/cresyl violet stain. Morphological analysis was performed on 12–15 sections per mouse, as previously described (19). Briefly, each quadrant from every coronal section from each mouse was graded for the presence or absence of gray matter disease, meningeal inflammation, and demyelination. The score was expressed as the percentage of spinal cord quadrants examined with the pathologic abnormality. The maximum score of 100 indicated that there was a particular pathologic abnormality in every quadrant of all spinal cord sections of a given mouse. All grading was performed without knowledge of the treatment group. To measure the actual areas (mm2) of demyelination and total white matter in the spinal cord cross sections, a Zeiss interactive digital analysis system (ZIDAS) and camera lucida attached to a Zeiss photomicroscope (Carl Zeiss, Thornwood, NY) was used, as previously described (20). Additional spinal cord blocks were embedded in paraffin for immunocytochemistry.
Immunostaining
Immunocytochemistry was performed on paraffin-embedded sections, as previously described (20). Slides were deparaffininized in xylene. Rehydration was performed through an ethanol series (absolute, 95%, 70%, 50%). Virus Ag staining was conducted using a polyclonal antisera to TMEV (DAV strain) that reacts strongly with the capsid proteins of TMEV. Staining for macrophages was performed using a rat anti-mouse Ab to F4/80 (Serotec, Raleigh, NC). Following incubation with a secondary biotinylated Ab (Vector Laboratories, Burlingame, CA), immunoreactivity was detected using the avidin-biotin immunoperoxidase technique (Vector Laboratories). The reaction was developed using Hanker-Yates reagent with hydrogen peroxide as the substrate (Polysciences, Warrington, PA). Slides were lightly counterstained with Mayer’s hemotoxylin.
Plaque reduction assay
The ability of rTGF-ß2 to directly inhibit Theiler’s virus plaque formation was determined by a plaque reduction assay, as previously described (9). L2 cell monolayers grown in 12-well tissue culture dishes (Costar) were either preincubated with rTGF-ß2 (0.1, 1, 10, 100 ng/ml) for 4 h before exposure to virus or were exposed to virus and cytokine simultaneously. Following virus exposure, the cell monolayers were overlaid with agarose (0.8%, Seaplaque; ICN Biochemicals). Cells were fixed and stained with 1% cresyl violet at 3 days p.i. All assays were performed in triplicate.
TMEV-specific ELISA
Blood was obtained from mice at the time of sacrifice and allowed to clot, and the sera were stored at -80°C until the time of assay. Polystyrene microtiter plates (Immunolon II; Dynatech, Chantilly VA) were coated with 0.5 µg of purified TMEV in 0.1 M carbonate buffer (pH 9.5), then blocked with 1% BSA (Sigma, St. Louis, MO) in PBS. Sera from individual mice were diluted in 0.2% BSA and incubated at room temperature. Biotinylated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) was used as the secondary Ab. Detection was performed using alkaline phosphatase-labeled streptavidin (Jackson ImmunoResearch), with p-nitrophenylphosphate used as the substrate. Absorbances were read at A405 (21).
Virus neutralization assay
Virus-neutralizing Abs were assessed as previously described
(22). Briefly, TMEV was diluted to contain 
200 PFU per
sample and then mixed with an equal volume of serial 2-fold dilutions
of heat-inactivated sera from infected mice. Following incubation on
ice for 30 min, this mixture was assayed for infectivity by plaque
assay on L2 cells.
Nonspecific T cell-mediated cytotoxicity assay
1452C11 cells (an anti-CD3 hybridoma cell line) were
cultured in RPMI 1640 (BioWhittaker, Walkerville, MD) with 5% FCS
(Life Technologies, Grand Island, NY). They were used as targets to
detect nonspecific T cell-mediated cytotoxicity. Activated cytotoxic
cells expressing CD3 bind anti-CD3 Abs on the 1452C11 cell surface
and lyse 1452C11 cells. On the day of assay, 1452C11 cells were labeled
with 200 µCi of 51Cr (Amersham Life Sciences,
Arlington Heights, IL), washed with RPMI, and resuspended to 2 x
104/ml in RPMI with 5% FCS. The target cell
suspensions (100 µl) were placed in 96-well round-bottom microtiter
plates (Nunc, Roskilde, Denmark). CNS-infiltrating lymphocytes
(CNS-ILs) from Theiler’s virus-infected mice were used as effector
cells in this assay. Seven days postinjection, the brains and spinal
cords from intracerebral infected SJL/J mice were pooled by strain or
treatment group. Mononuclear cells were isolated, as described
previously (23). Briefly, brains and spinal cords were
homogenized with a tissue grinder, and the mononuclear cells were
isolated over a Percoll (Pharmacia Biotech, Piscataway, NJ) gradient by
centrifugation at 27,000 x g. The mononuclear cell
band was removed, washed, precleared of erythrocytes with sterile
distilled H2O, and counted. The CNS-ILs were
resuspended to 2 x 106/ml in RPMI with 5%
FCS, and 2-fold serial dilutions were made to provide E:T ratios of
100:1 to 6.25:1. The effector cell suspensions (100 µl) were added in
triplicate to the targets, resulting in final E:T ratios of 100, 50,
25, 12.5, and 6.25 to 1. Six wells of targets also received medium
alone or 10% Triton X-100 (Sigma) to determine spontaneous release and
maximum release of chromium from targets, respectively. Plates were
incubated for 5 h at 37°C in 5% CO2.
Supernatants were harvested with Skatron Supernatant collection system
(Skatron Instruments, Sterling, VA) and assayed in a gamma counter
(Beckman 5500; Beckman Instruments, Irvine, CA) for the amount of
radioactivity. Mean values were calculated from triplicate wells, and
results were expressed as percent specific lysis according to the
formula: [(experimental counts - spontaneous counts)/(maximum
counts - spontaneous counts)] x 100%. The SEM was determined
from the results obtained from pooled lymphocyte samples in triplicate
wells. Statistical comparisons were performed by using the unpaired
Student’s t test.
Virus-specific T cell-mediated cytotoxicity
CTL assays were performed as described above using TMEV-infected KSSV (KsDs) target cells (23). Uninfected KSSV cells were used as controls.
TUNEL assay
TUNEL was performed on paraffin-embedded slides using the ApopTag peroxidase in situ apoptosis detection kit per the manufacturer’s protocol (Intergen, Purchase, NY). Briefly, slides were deparaffinized in xylene, then rehydrated through an alcohol series (100%, 95%, 70%, 50%, 30%) before rehydrating in PBS. The slides were postfixed in ethanol:acetic acid (2:1) at -20°C, washed in PBS, then incubated in equilibration buffer at room temperature before incubation with digoxigenin-labeled TdT at 37°C. After washing, slides were incubated with peroxidase-labeled anti-digoxigenin Ab, then developed with Hanker-Yates reagent (Polysciences, Warrington, PA), with hydrogen peroxide as the substrate.
Statistics
Statistical analyses were performed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first addressed whether or not TGF-ß2 treatment would
influence the frequency or extent of demyelinating lesions in SJL/J
mice infected with TMEV. Mice were treated three times per week with
either 0.1 µg of TGF-ß2 i.p. beginning on the day before infection,
or 1 µg of TGF-ß2 beginning at day 15 p.i. Quantitative
morphology was performed in the spinal cord to determine the extent of
disease observed at day 35 p.i. Previous studies from our
laboratory have demonstrated that this time point can effectively
distinguish between mouse strains susceptible or resistant to
TMEV-induced demyelinating disease (24, 25, 26). Treatment
with TGF-ß2 did not affect the neuronal (gray matter) disease (Table I
). Following administration of either a
high dose (beginning at day +15 p.i.) or low dose (beginning at day
-1) of TGF-ß2, the incidence of demyelinating lesions in the spinal
cord was significantly reduced as compared with infected control mice
(Fig. 1, A and B;
Table I; p = 0.04). To test whether TGF-ß2 also
affected lesion size, we measured the lesion area using a Zeiss
photomicroscope with a Zidas interactive camera lucida system. The
lesions in the TGF-ß2-treated animals were significantly (60–70%)
smaller than those observed in the control-treated mice (Table II; p = 0.03). Together,
these results conclusively demonstrate that TGF-ß2 treatment reduces
both the incidence and size of lesions formed following infection
with TMEV.
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Previous studies using this model have demonstrated that the
extent of demyelination correlated with levels of virus Ag and RNA in
the spinal cord (27). To determine whether TGF-ß2
reduced demyelination via a mechanism involving a reduction of viral
load, immunostaining was performed on paraffin-embedded spinal cord
blocks using a polyclonal antisera to the capsid proteins of Theiler’s
virus. Total white matter area measurements were made using a Zeiss
Image Analysis system attached to an Axiophot microscope, and the data
were expressed as the number of virus Ag-positive
cells/mm2 of white matter. As shown in Table III, mice administered TGF-ß2 beginning
at day 15 p.i. had significantly fewer TMEV-positive
cells/mm2 white matter as compared with infected
control-treated mice (p = 0.03; Fig. 1
, C and D). Mice treated with a lower dose of
TGF-ß2 beginning 1 day before infection also had fewer TMEV-positive
cells compared with those of the PEG group, but did not reach the level
of statistical significance (p = 0.06). These
results suggest that TGF-ß2 may influence the level of demyelination
following TMEV infection by directly or indirectly controlling virus in
the white matter of the spinal cord.
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As TMEV Ag levels were reduced following in vivo treatment with TGF-ß2, we tested whether TGF-ß2 would reduce replication of TMEV in vitro. L2 cells were treated with 100, 10, 1, or 0.1 ng/ml of TGF-ß2 or media for 4 h before addition of 50 PFUs of TMEV. Similar numbers of plaques were observed in all treatment groups (41.3 ± 9.8, 37.4 ± 3.2, 35.9 ± 7.3, 40.6 ± 2.1, 37.8 ± 6.9 plaques per well for 100, 10, 1, 0.1, and 0 ng/ml of TGF-ß2, respectively), demonstrating no detectable effects of TGF-ß2 treatment on TMEV replication in vitro. Similar results were obtained with simultaneous addition of virus and TGF-ß2 (data not shown). These experiments demonstrate that it is unlikely that TGF-ß2 reduces viral burden by directly inhibiting viral replication.
TGF-ß2-treated mice developed normal humoral immune responses to TMEV
Studies from multiple laboratories have indicated that Igs may
influence the level of demyelination resulting from TMEV infection of
susceptible strains of mice (28, 29). To determine whether
TGF-ß2 treatment affected Ab production, TMEV-specific IgG levels
were measured by ELISA using purified TMEV Ags. TGF-ß2 and
vehicle-treated infected mice developed similar anti-TMEV IgG
levels (Fig. 2). One possibility is that
TGF-ß2 treatment altered the levels of neutralizing Ab in the sera
without affecting the level of total virus-specific IgG. To test
whether TGF-ß2-treated mice had increased levels of TMEV-specific
neutralizing Ab compared with vehicle-treated infected mice,
virus-neutralization assays were performed. No significant differences
in mean number of plaques per treatment were found between groups (PEG,
23.7 ± 0.9 plaques; TGF-ß2, day +15, 27.3 ± 4.2;
TGF-ß2, day -1, 28.5 ± 15.7). Together, these data demonstrate
that TGF-ß2 does not reduce demyelination by modulating the
TMEV-specific humoral immune response.
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Although TGF-ß2 is generally considered to be immunosuppressive,
Kondo et al. (30) demonstrated that in vitro treatment of
murine T cells with TGF-ß significantly enhanced the generation of
allospecific cytotoxic T cells. As previous studies from our laboratory
(31) and others (32) have demonstrated that
CTLs are critical to protection from TMEV-induced demyelinating
disease, we tested whether in vivo treatment with TGF-ß2 altered the
level of cytotoxicity observed in the CNS of TMEV-infected mice. In
this study, SJL/J mice were treated with either TGF-ß or vehicle at
days -1, +1, +3, and +5. At day 7 p.i., mice were sacrificed and
the CNS-ILs were tested for TMEV-specific cytotoxicity using
TMEV-infected KSSV (KsDs)
target cells. No TMEV-specific CTL activity was detected in either
TGF-ß2- or vehicle-treated SJL/J mice (Fig. 3), consistent with the previous
observation that mice susceptible to demyelination fail to generate
virus-specific CTLs in the CNS (33).
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; p <
0.05, comparing percent lysis at the 100:1, 25:1, 12.5:1, and 6.25:1
CNS-ILs to target ratio). TGF-ß2 reduces apoptosis in the spinal cord white matter of TMEV-infected mice
Several studies have demonstrated that TGF-ß2 can alter
apoptotic processes in CNS resident cells (34, 35). To
test whether the mechanism by which TGF-ß2 reduces spinal cord
pathology in TMEV-infected mice by changing the level of apoptosis in
the white matter, a TUNEL assay was performed on spinal cord sections
from mice at day 35 p.i. TUNEL-positive cells were detected
throughout the white matter (both within and outside of the lesion
area) of both TGF-ß2 (1 µg at day +15, n = 6)- and
control (n = 5)-treated mice (Fig. 4, A and B).
Significantly more apoptotic cells/mm2 of spinal
cord white matter were detected in the white matter of vehicle-treated
infected mice than the TGF-ß2-treated mice (10.4 ± 2.3
TUNEL-positive cells/mm2 white matter and
4.6 ± 1.1 cells/mm2, respectively;
p = 0.03). The TUNEL-positive cells included both
resident and infiltrating cells; thus, at least part of the observed
difference could be attributed to the degree of inflammation observed
in the different groups.
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Several studies have implicated macrophages as a reservoir of virus in TMEV-infected mice (36, 37). Depletion of macrophages with liposomes has been shown to reduce demyelination in susceptible mouse strains by preventing virus persistence (37). TGF-ß2 has been demonstrated to decrease macrophage activation (38, 39, 40, 41). To determine whether TGF-ß2 could reduce TMEV Ag levels and subsequent demyelinating sequelae via an alteration in macrophage recruitment, immunostaining was performed on spinal cord sections from SJL/J mice at day 35 p.i. The data were expressed as the number of F4/80-positive cells/mm2 of spinal cord white matter. Immunostaining revealed increased numbers of macrophages in control-treated infected mice (27.7 macrophages/mm2 of spinal cord white matter) as compared with TGF-ß2-treated (1 µg beginning at day 15 p.i.) infected mice (7.3 macrophages/mm2 white matter; p = 0.01). These data suggest that one potential mechanism that may account for the reduction in virus burden and demyelination may be related to a decrease in macrophages in the spinal cord of TGF-ß2-treated mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In view of the results of the plaque reduction assay, we examined several mechanisms by which TGF-ß2 could indirectly affect virus burden. To determine whether TGF-ß2 altered virus-specific humoral immune responses, we measured the levels of both total TMEV-specific IgG by ELISA and TMEV-specific neutralizing Ab by plaque reduction assay. We found no difference between the treated and the control mice, suggesting that the mechanism through which TGF-ß2 reduces demyelination does not involve modulation of the humoral immune response.
Based on its ability to decrease T and B cell proliferation, suppress cytokine production, and reduce macrophage activation, TGF-ß2 is considered to be an immunosuppressive cytokine. In addition, TGF-ß2 has also been shown to depress NK cell activity and CTL generation. These responses are not universal, however, in that Kondo et al. (30) have demonstrated that in vitro TGF-ß2 can enhance allospecific CTL generation. Several studies from our laboratory (23, 31, 43) and others (32) have demonstrated that resistance to TMEV-induced demyelination is related to the ability to mount a CTL response to the VP2-specific region of TMEV in the CNS (30). The role of CD8+ T cells in SJL/J mice appears to be more complex than the situation observed in mice on a C57BL background. Studies in SJL/J mice utilizing Abs to deplete CD8+ T cells demonstrated that a reduction in CD8+ T cells led to a decrease in demyelination (5). In contrast, genetic deletion studies in SJL/J mice demonstrated that CD8+ T cells did not influence demyelination (44). To test whether TGF-ß2 increased CTL activity in the CNS of TMEV-infected mice, we assessed CTL activity day 7 p.i. No increase in TMEV-specific CTLs was observed, although CTL activity against nonviral Ags was enhanced. Because of the lack of virus-specific CD8+ in SJL/J mice, any protective role that these nonspecific CTLs would play in this system would likely be via an indirect regulatory role. These activated CTLs without apparent virus specificity may participate to control virus infection via the release of cytokines, as has been reported in other systems (45).
TGF-ß2 has been reported to down-regulate macrophage function (38, 40, 41). This effect of TGF-ß2 is particularly interesting when previous studies addressing the role of macrophages in TMEV infection are considered. These studies have suggested that macrophages are an important reservoir of TMEV during the chronic stages of disease, and depletion of macrophages prevents chronic demyelination from commencing (37). We hypothesize that the most likely scenario that accounts for the reduction in demyelination is that TGF-ß2 decreases macrophage/microglial function or infiltration to the CNS, thereby directly decreasing viral load and subsequent demyelination. This provides a mechanism of protection from demyelination that is unique from that described in the EAE studies and supports a potential role for TGF-ß in therapy for macrophage-mediated diseases in which an infectious agent resides in these cells.
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Footnotes |
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2 Current address: Department of Medical Microbiology and Immunology, Creighton University, Criss I, Room 521, 2500 California Plaza, Omaha, NE 68178.
3 Current address: Department of Neurology, University of Chicago Medical Center, MC 2030, 5841 S. Maryland Avenue, Chicago, IL 60637
4 Address correspondence and reprint requests to Dr. Moses Rodriguez, Department of Immunology, Mayo Clinic/Foundation, 200 First Street, S.W., Guggenheim (4), Rochester, MN 55905. E-mail address:
5 Abbreviations used in this paper: TMEV, Theiler’s murine encephalomyelitis virus; CNS-IL, CNS-infiltrating lymphocyte; EAE, experimental autoimmune encephalomyelitis; PEG, polyethylene glycol; p.i., postinfection.
Received for publication October 25, 1999. Accepted for publication January 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interferon is critical for resistance to Theiler’s virus-induced demyelination. J. Virol. 69:7286.[Abstract]
/ß or interferon receptors. J. Exp. Med. 181:2069.. Int. Immunol. 2:909.. J. Immunol. 145:940.[Abstract]
exerts an antiviral effect in the central nervous system but not in peripheral solid organs. J. Immunol. 150:2316.[Abstract]
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