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Cutting Edge |
Dependent in Mice Infected with a Neurotropic Coronavirus1
Departments of Pediatrics and Microbiology, 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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-/- donors resulted in
a substantial decrease in demyelination (4.8% of the white matter of
the spinal cord compared with 26.3% in those receiving cells from
C57BL/6 donors). Similar numbers of lymphocytes were present in the CNS
of recipients of either C57BL/6 or IFN--/- CD8 T
cells, suggesting that IFN- was not crucial for lymphocyte entry
into the CNS. Rather, IFN- was critical for optimal activation or
migration of macrophages or microglia into the white matter in the
context of CD8 T cell-mediated demyelination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice infected with the JHM strain of mouse hepatitis virus (MHV)
develop a fatal encephalitis, unless the infection is modified by
treatment with antivirus Abs or T cells or by infection with attenuated
strains (7, 8). One such strain, MHV 2.2-V-1, causes
demyelination in immunocompetent C57BL/6 (B6) mice with less
mortality than in the parental strain. When SCID or recombination
activation gene 1-deficient (RAG1)-/- mice
were infected with MHV 2.2-V-1, they succumbed to encephalitis 12–14
days postinoculation without evidence of significant demyelination in
their spinal cords. Adoptive transfer of splenocytes from B6 mice into
infected SCID or RAG1-/- mice resulted in the
appearance of demyelination 
7 days post-transfer (p.t.) (9, 10). The development of demyelination was most consistent in
RAG1-/- mice when splenocytes were transferred
from donors previously immunized with MHV (10). Transfer
of cells from naive donors only sporadically resulted in demyelination
in this model.
In subsequent experiments, we showed that depletion of both CD4 and CD8
T cells from the donor populations abrogated the appearance of
demyelination whereas depletion of only one T cell subset did not.
Recipients of CD4 T cell-enriched populations developed severe
encephalitis (hunching, lethargy, ruffled fur) 6–7 days p.t., whereas
recipients of CD8 T cells remained largely asymptomatic until 7–10
days p.t., when they developed signs of hind limb weakness. More
extensive demyelination developed in recipients of CD8 T cell-enriched
populations (5). Little is known about the effector
molecules important for CD4 or CD8 T cell-mediated demyelination in
this model. Previous studies indicated that no single effector molecule
was required for MHV-induced demyelination. Perforin, TNF-
, IFN-,
inducible nitric oxide synthase, and IL-10 were studied in these
reports (8).
A conclusion from one study was that IFN-, a key proinflammatory
molecule, was critical for virus clearance from infected
oligodendrocytes, although not for the development of demyelination
(11). Overexpression of IFN- has also been shown to
enhance recruitment of cells to the MHV-infected CNS, perhaps via
activation of endothelial cells (12). Recent studies
indicate that IFN- also has a suppressive role in CNS inflammatory
disease and in chemokine regulation (13). Because CD4 or
CD8 T cell-enriched populations were able to cause demyelination but
with different patterns of clinical disease, we reasoned that each
subset might cause demyelination using a different set of effector
molecules. For these reasons, we decided to reinvestigate the roles of
IFN-, perforin, and TNF- in MHV-induced demyelination in the
context of CD8 T cell enrichment. In this report, we show that
demyelination is largely abrogated when CD8 T cells from
IFN--/- mice are transferred.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The neuroattenuated variant of the JHM strain of MHV, MHV 2.2-V-1, generously provided by Dr. J. Fleming (University of Wisconsin, Madison, WI), was used in all studies.
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) and bred at
the University of Iowa (Ames, IA). IFN--/-
mice were obtained from The Jackson Laboratory.
Adoptive transfer model
RAG1-/- mice were infected with
1 x 103 PFU MHV 2.2-V-1 by intracranial
injection. Adoptive transfer of 5 x 106
splenocytes from B6 or IFN--/- mice
immunized i.p. with wild-type MHV to infected
RAG1-/- mice was performed as previously
described (10). Wild-type MHV was used for immunization to
maximize the anti-MHV immune response in donor animals. A total of
85 MHV 2.2-V-1-infected RAG1-/- mice were used
in these experiments; 35 received B6, 25 received
IFN--/-, 18 received
perforin-/-, 3 received
TNF--/-, and 4 received
TNF--perforin-/- CD8 T cell-enriched
splenocytes. No infectious virus could be detected by plaque assay in
the transferred cells (10).
Complement depletion
Donor splenocytes depleted of CD4 T cells were prepared for adoptive transfer by two rounds of complement lysis, as previously described (5). Under these conditions, depletion was >98% and no MHV-specific CD4 T cells were detected in the CNS of infected recipient mice at 10 days p.t. (5).
Immunohistochemistry
Sections were stained for macrophages/microglia with rat anti-F4/80 mAb (CI:A3–1; Serotec, Oxford, U.K.) as previously described (5).
Flow cytometry
Lymphocytes were prepared from the CNS as described previously (14). Briefly, single-cell suspensions were blocked with purified anti-mouse CD16/CD32 (mAb 2.4G2) (BD PharMingen, San Diego, CA) in 10% rat serum. For two- or three-color flow cytometric analysis, cells were stained with combinations of the following Abs: FITC-conjugated rat anti-mouse CD8, CD45 (BD PharMingen); PE-conjugated rat anti-mouse CD11b (Mac-1; BD PharMingen); and biotinylated anti-CD8 Ab. Biotinylated Ab was detected by avidin-APC (Molecular Probes, Eugene, OR). In all cases, an isotype-matched FITC- or PE-conjugated Ab was used. Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA) or an EPICS 753 (Beckman Coulter, Fullerton, CA) at the University of Iowa FACS facility (Ames, IA).
Intracellular staining for IFN- or TNF-
Lymphocytes were prepared from the CNS and stimulated with
peptide-coated EL-4 cells for Ag presentation to CD8 T cells in the
presence of brefeldin A or monensin (Golgiplug or Golgistop; BD
PharMingen). Peptides corresponding to the immunodominant CD8 T cell
epitope recognized in B6 mice (residues 510–518 of the surface
glycoprotein (S510)) (15, 16) or
OVA257–264 (OVA257,
irrelevant peptide) were used at a final concentration of 1 µM. Cells
were processed for IFN- or TNF- expression as previously
described (5). Lymphocytes harvested from CNS tissue of
single mice or pools of two to four mice were used in these analyses.
The absolute number of Ag-specific cells was calculated by multiplying
the fraction of Ag-specific CD8 T cells by the fraction of CD8 T
lymphocytes by the total number of cells per brain.
Tetramers
Tetramers of MHC class I (H-2Db) peptide S510 conjugated with avidin-PE (Molecular Probes) were obtained from the National Institute of Allergy and Infectious Diseases Tetramer Core Facility, Atlanta, GA.
Imaging and quantification of demyelination
Demyelination was quantified using Vtrace software (Image Analysis Facility, University of Iowa, Ames, IA), as previously described (5).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- CD8 T cell-enriched splenocytes
MHV-infected RAG1-/- mice develop
clinical disease consisting of lethargy, ruffled fur, wobbly gait, and
hind limb paresis 7–9 days after adoptive transfer of undepleted
MHV-immune B6 or IFN--/- splenocytes.
Examination of spinal cords revealed 15–20% demyelination of the
white matter of the spinal cord in recipients of either B6 or
IFN--/- cells (data not shown).
In contrast, greater morbidity was observed in recipients of
IFN--/- CD8 T cell-enriched splenocytes than
in those receiving similar populations from B6 mice. We showed
previously that MHV-infected recipients of B6 CD8 T cell-enriched
splenocytes developed mild disease characterized by hind limb
paresis/paralysis with few signs of encephalitis (5). Mice
often survived to day 15 p.t. or longer. In contrast, recipients
of IFN--/- CD8 T cell-enriched splenocytes
showed clinical signs of encephalitis (lethargy, ruffled fur, wasting,
hunching) by 9–10 days p.t. and did not survive past 12 days p.t. Limb
weakness, observed in mice with extensive demyelination, was not a
prominent feature of disease in these animals. Virus titers were
similar in recipients of CD8 T cells from B6 or
IFN--/- mice (Table I
), making it unlikely that inefficient
virus clearance in recipients of IFN--/-
cells accounted for the observed differences in clinical disease.
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-/- CD8 T cells exhibited significantly
less demyelination when compared with those receiving cells from B6
mice (Table I
and Fig. 1, A
and C). Ten days after transfer of B6 CD8 T cell-enriched
splenocytes, 26.3 ± 6.0% of the white matter of the spinal cord
showed evidence of demyelination with extensive macrophage infiltration
(Fig. 1B). In contrast, demyelination was markedly reduced
in recipients of IFN--/- CD8 T cell-enriched
splenocytes, encompassing, on the average, 4.8 ± 1.3% of the
white matter. Most strikingly, we detected equal or greater numbers of
activated macrophages/microglia in the gray matter, but greatly reduced
numbers in the white matter, in recipients of
IFN--/- cells when compared with mice
receiving B6 splenocytes (Fig. 1, B and D;
quantification is described below). The amount of demyelination was
greater in recipients of IFN--/- cells than
in infected RAG1-/- mice in the absence
of adoptive transfer in which only 1–2% of the spinal cord white
matter showed evidence of myelin destruction (10). Thus,
donor cell-derived IFN- was critical, but not absolutely required,
for CD8 T cell-mediated demyelination.
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or whether
any defect in CD8 T cell effector function would affect demyelination,
CD8 T cell-enriched populations from
perforin-/-,
TNF--/-, or
perforin-TNF--/- donors were transferred to
MHV-infected RAG1-/- mice (Table I). Less
demyelination was observed in recipients of
perforin-/- CD8 T cell-enriched splenocytes
(15.5% ± 2.1) than in recipients of B6 cells (26.3% ± 6.0), but the
difference did not reach statistical significance
(p = 0.07). At least as much demyelination was
detected in recipients of TNF--/- CD8 T
cells as in those receiving B6 cells. As in recipients of
perforin-/- CD8 T cell-enriched populations,
17.8 ± 2.3% of the white matter of the spinal cord was
demyelinated in recipients of cells from
perforin-TNF--/- donors. Thus, perforin may
have a role in demyelination, but its absence does not have as profound
an effect as the absence of IFN- in the donor cell population.
Infiltration of CD8 T cells is the same in recipients of B6 or
IFN--/- CD8 T cells
One explanation for these results is that in the absence of
IFN- in donor cells, recruitment of CD8 T cells or other
inflammatory cells is inefficient. Our immunohistochemical analyses
suggested that macrophage/microglia (Fig. 1, B and
D), but not CD8 T cell (data not shown), infiltration into
the white matter was diminished in recipients of
IFN--/- cells as compared with those
receiving B6 cells. To quantify these results more precisely, we
analyzed CNS-derived mononuclear populations for CD8 T cells and
macrophages/microglia (Table II). The
number of MHV-specific CD8 T cells in the CNS was assessed using
S510 tetramers. As shown in Table II,
similar numbers of tetramer S510-positive CD8 T
cell lymphocytes were detected in the CNS in recipients of either
IFN--/- or B6 donor cells.
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expression after peptide S510 stimulation in
recipients of B6 or IFN--/- splenocytes and
IFN- expression in recipients of B6 donor cells. In control
experiments, we showed that assays for IFN- or TNF- identified
nearly identical numbers of epitope S510-specific
T cells in the CNS of recipients of undepleted B6 populations (IFN-
16.7 ± 2.4% vs TNF- 15.4 ± 2.2%, n =
7). Consequently, data from experiments in which epitope
S510-specific T cells were detected with either
cytokine are included in Table II. In other experimental systems, more
pathogen-specific primary CD8 T cells expressed IFN- than TNF-
after peptide stimulation directly ex vivo, but memory cells expressed
both cytokines. After secondary infection, most CD8 T cells expressed
both cytokines (17, 18). These results suggest that after
immunization and adoptive transfer, CD8 T lymphocytes harvested from
the CNS of recipient mice function as secondary effector cells. Only a
fraction of cells secreted IFN- or TNF- in response to peptide
S510, suggesting that in both cases, only a
minority of cells were capable of cytokine effector function. This
number was, however, similar in recipients of B6 or
IFN--/- CD8 T cell-enriched splenocytes.
Decreased macrophage/microglia infiltration into the CNS of
recipients of IFN--/- CD8 T cells
In contrast, macrophage/microglia staining and FACS analysis
revealed a decrease in the number of activated
CD11b+CD45high cells in the
CNS of recipients of IFN--/- cells when
compared with those receiving cells from B6 donors (B6, 3.6 ±
1.0 x 104;
IFN--/-, 2.0 ± 0.3 x
104; Table II) although these differences were
not statistically significant (p = 0.10). In
another approach, we counted the number of macrophages/microglia in the
gray and white matter of sagittal sections of spinal cords from three
mice that received B6 CD8 T cell-enriched splenocytes and three that
received IFN--/- cells. For this purpose,
all of the F4/80+ cells in 1.25-mm-wide
cross-sections at eight levels within spinal cords were counted.
Consistent with the FACS analysis, the number of macrophages/microglia
present in each cross-sectional area in recipients of
IFN--/- CD8 T cells was decreased by 45%
(B6 vs IFN--/-, 179.4 ± 9.7 vs
80.3 ± 7.4, p < 0.0001). Furthermore, there was
a 6- to 7-fold reduction in the number of F4/80+
cells in the white matter (B6 vs IFN--/-,
145.2 ± 10.1 vs 21.9 ± 4.0, p < 0.0001)
but more cells in the gray matter (B6 vs
IFN--/-, 34.1 ± 3.9 vs 58.4 ±
5.5, p < 0.001) of recipients of
IFN--/- cells than in that of those
receiving cells from B6 donors. These results suggest that CD8 T
cell-derived IFN- is important in both macrophage/microglia
activation and in migration of these cells into the white matter of the
spinal cord.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is critical for CD8 T cell-mediated demyelination, in agreement with
the conclusions of Huseby et al. (1). Furthermore, our
data show that IFN- is important for macrophage/microglia activation
or migration into the white matter of the CNS in the absence of CD4 T
cells but is not required for the migration of CD8 T cells into the
CNS. Demyelination was not reduced when undepleted
IFN--/- splenocytes were transferred, in
agreement with previous results (11), nor is demyelination
diminished in recipients of IFN--/- CD4 T
cells when compared with those receiving B6 cells (data not shown).
IFN- has multiple effects in CNS inflammation, both as a
proinflammatory molecule and in regulating leukocyte trafficking and T
cell population dynamics (21, 22, 23, 24). In its proinflammatory
role, it is involved in such functions as macrophage activation,
up-regulation of MHC class I and class II expression, and induction of
adhesion molecules important for entry into the CNS. As a
proinflammatory mediator, IFN- is involved in the induction of
expression of chemokines, such as C-C chemokine ligand (CCL)
2/macrophage chemoattractant protein (MCP)-1, CCL7/MCP-3,
CCL4/macrophage-inflammatory protein-1
, CCL5/RANTES, CXC ligand
9/monokine induced by IFN- and CXC ligand 10/IFN-inducible protein
10/cytokine-responsive gene 2, which are chemoattractants for
monocytes/macrophages and lymphocytes and are up-regulated in the CNS
of MHV-infected mice (25). To determine whether reduced
expression of any of these chemokines might be involved in the
diminished macrophage/microglia activation or migration observed after
transfer of IFN--/- CD8 T cells, we assayed
CNS samples for chemokine RNA expression. We found that each of these
chemokines was expressed at equivalent or higher levels in recipients
of IFN--/- CD8 T cells than in those
receiving B6 CD8 T cells (data not shown). This indicated that none was
sufficient for macrophage/microglia infiltration or activation.
In summary, we showed that IFN- is critical for CD8 T cell-mediated
demyelination in MHV-infected mice, most likely by facilitating
macrophage activation or migration into the CNS. Our results may be
relevant for understanding the human disease MS, because administration
of recombinant IFN- worsened clinical disease in patients
(26). This model system should be useful for delineating
the precise mechanism of action of IFN- in the demyelinating
process.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stanley Perlman, Medical Laboratories 2042, Department of Pediatrics, University of Iowa, Iowa City, IA 52242, E-mail address: Stanley-Perlman{at}uiowa.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MHV, mouse hepatitis virus; MCP, macrophage chemoattractant protein; RAG1, recombination activation gene 1; p.t., post-transfer; S510, residues 510–518 of the surface glycoprotein; CCL, C-C chemokine ligand.
Received for publication November 6, 2001. Accepted for publication December 19, 2001.
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References |
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is required for viral clearance from central nervous system oligodendroglia. J. Immunol. 162:1641. by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread and pathogenicity. Virology 233:327.[Medline]
detects different frequencies of antigen-specific CD8+ T cells. J. Immunol. Methods 238:107.[Medline]
in immune cell regulation. J. Leukocyte Biol. 58:373.[Abstract]
plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759.-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123.- and -chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160:970. interferon. Lancet 1:893.[Medline]This article has been cited by other articles:
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C. Savarin, C. C. Bergmann, D. R. Hinton, R. M. Ransohoff, and S. A. Stohlman Memory CD4+ T-Cell-Mediated Protection from Lethal Coronavirus Encephalomyelitis J. Virol., December 15, 2008; 82(24): 12432 - 12440. [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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M. Brisebois, S. P. Zehntner, J. Estrada, T. Owens, and S. Fournier A Pathogenic Role for CD8+ T Cells in a Spontaneous Model of Demyelinating Disease J. Immunol., August 15, 2006; 177(4): 2403 - 2411. [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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 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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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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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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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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