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Is Required for Viral Clearance from Central Nervous System Oligodendroglia1
,
Departments of
*
Molecular Microbiology and Immunology,
Neurology, and
Pathology, University of Southern California School of Medicine, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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as
mediator controlling JHMV replication in oligodendroglia was examined
in mice deficient in IFN- secretion (IFN-0/0 mice).
IFN-0/0 mice exhibited increased clinical symptoms and
mortality associated with persistent virus, demonstrating an inability
to control replication. Neither antiviral Ab nor CTL responses were
diminished in the absence of IFN-, although increased IgG1 was
detected in IFN-0/0 mice. Increased virus Ag in the
absence of IFN- localized almost exclusively to oligodendroglia and
was associated with increased CD8+ T cells localized within
white matter. These data suggest that although perforin-dependent CTL
control virus replication within astrocytes and microglia, which
constitute the majority of infected CNS cells, IFN- is critical for
control of viral replication in oligodendroglia. Therefore, different
mechanisms are used by the host defenses to control virus replication
within the CNS, dependent upon the phenotype of the targets of virus
replication. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß (1). CNS
viral infections result in vigorous immune responses; however, these
inherent limitations appear to contribute to viral persistence or
latency (2). Indeed, infection by a variety of both RNA and DNA viruses
ultimately results in persistent CNS infections. Although many viruses
predominantly infect single CNS cell types, each of the major cell
types can provide a reservoir for persistent or latent viral infection.
For example, herpes simplex virus and measles virus infect and
subsequently persist in neurons (3, 4). Other viruses, such as
lymphocytic choriomeningitis virus (LCMV) and the neurotropic
coronavirus strain JHM (JHMV) initially infect a variety of cell types
(5, 6, 7, 8) but subsequently persist in only a subset of CNS cells (9, 10, 11, 12).
Immunity within the CNS appears to arrive at a balance that eliminates
infectious virions and minimizes damage by either allowing or actually
facilitating viral persistence. A remaining unanswered question is how
immune effector mechanisms influence viral infections of the diverse
cell types that comprise the CNS, resulting in persistent infection
within specific cell types. Infection of the rodent CNS by JHMV provides a model of acute viral infection that progresses to a chronic infection associated with ongoing CNS demyelination (9, 13, 14). The immune response contributes to both viral clearance (15, 16), but also to viral-induced primary demyelination (13, 17). Virus-specific Ab and T cell responses (15, 16, 18) have been implicated in limiting infectious virus. Although these immune effector mechanisms suppress virus replication, they are unable to completely eliminate virus resulting in persistent infection (19). JHMV replication in oligodendroglia may contribute to demyelination (8); however, increasing evidence suggests that demyelination is immunopathologically mediated and possibly distinct from the immune mechanisms that control virus replication (13, 14, 17). Defining the roles of the immune effectors that contribute to elimination of infectious virus is clarifying the strategies of JHMV persistence within the CNS. Neutralizing Ab are generally detected only after JHMV clearance (20, 21) and are suspected of playing a role in establishing or maintaining chronic infection without directly contributing to viral clearance. By contrast, both CD4+ and CD8+ T cells accumulate within the CNS during acute JHMV infection and are associated with viral clearance (20, 22, 23). Virus-specific CD4+ T cells may (15) or may not (24) contribute directly to viral clearance, possibly via the secretion of soluble mediators. The CTL response limits virus replication in astrocytes and microglia but only to a lesser extent in oligodendrocytes (16) via a perforin-dependent mechanism (20). Indeed, CTL escape mutants of JHMV are associated with persistent CNS infectious virus (25, 26). The CTL-mediated reduction of virus replication in oligodendroglia was suggested to be a consequence of the overall successful CTL-mediated elimination of virus producing cells and not due to direct cytolysis of oligodendroglia (16).
IFN- plays important roles in many antiviral immune responses and is
predominantly secreted by CD8+ T cells (27, 28). Therefore,
the reduction of JHMV-infected oligodendrocytes in perforin-deficient
mice (20) could have occurred via IFN- secretion. Consistent with
this interpretation, inhibition of IFN- that enhances the severity
of viral infections (29, 30, 31, 32, 33, 34) is not correlated with defects in the
generation of other antiviral immune effectors (33, 34). The notion
that IFN- plays a critical role in the pathogenesis of JHMV with
tropism for microglia, astrocytes, and oligodendroglia is suggested by
several observations. Analysis of the kinetics of cytokine mRNA
accumulation within the CNS of mice infected with JHMV, including
infection with a neuronotropic variant of JHMV (OBLV-60), suggested a
relationship between IFN- and inhibition of viral replication (21, 35). T cell-dependent IFN- inhibits vaccinia virus replication in
meninges but not peripheral organs (36), suggesting that IFN-
secretion within the CNS may be a critical component in controlling
virus replication within specific CNS cell types. However, as shown by
the clearance of the OBLV-60 variant of JHMV from the CNS of
IFN-o/o mice (7), IFN- is not critical for virus
clearance from neurons. An antiviral role of IFN- is also supported
by the elimination of JHMV from the CNS of perforin-deficient mice (20)
and after adoptive transfer of either JHMV-specific CD4+ T
or CD8+ T cells (15, 16). Virus-specific T cells all
reduced virus in infected CNS cells including oligodendrocytes
potentially via secretion of IFN- (15, 16). Finally, the expression
of the IFN- receptor (37), but not MHC molecules on oligodendroglia
(2), suggest that IFN- may reduce JHMV replication in this cell
type. These observations provided the basis for examining the potential
contribution of IFN- in limiting JHMV infection of oligodendroglia.
Infectious virus was not completely cleared from the CNS of
IFN-0/0 mice, although CTL and neutralizing Ab responses
were induced. Consistent with a vigorous but ineffective CTL response,
CD8+ T cells were selectively recruited to white matter
areas of the CNS. Survivors showed persistence of viral Ag in
oligodendroglia consistent with CTL-mediated clearance from astrocytes
and microglia, but not oligodendrocytes (16). These data suggest that
although IFN- is not required for the inhibition of replication in
neurons (7), it either directly or indirectly controls viral
replication within oligodendroglia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice heterozygous for a nonfunctional IFN- gene
(IFN-0/0) were obtained at N9 from Genentech (San
Francisco, CA) (38) and maintained by homozygote mating. Syngeneic
wild-type (wt) C57BL/6 mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). Male and female 6–8-wk-old IFN-0/0
and wt mice were used in all experiments. Mice were infected by
intracerebral injection with 25 pfu of the 2.2v-1 mAb derived
neutralization escape variant of JHMV (39) and monitored for 21 days
postinfection (p.i.). This strain produces a subacute encephalomyelitis
with primary CNS demyelination and paralysis that progresses to chronic
infection (13, 19, 39). Clinical scores were read daily and graded as
described previously (20, 21, 39).
Virus titration
Brains were divided into thirds in the sagittal plane and processed for virus titration, histopathology, and RNA extraction. Viral titers were determined by plaque assay on monolayers of DBT cells as described previously (16, 20, 21). Briefly, tissue was homogenized in 4 ml of Dulbecco’s PBS (pH 7.4) and centrifuged (1500 x g for 7 min at 4°C), and supernatants assayed immediately or stored at -70°C. Average titers of groups of at least three mice per time point are presented.
Virus-specific Ab response
JHMV-specific IgG1 and IgG2a serum Ab were quantitated by ELISA as described (20, 21) and expressed as the log of the highest dilution with OD values three times above background level. Rabbit anti-mouse IgG1 or IgG2a were used as secondary Ab (Cappel Laboratories, Costa Mesa, CA). Neutralizing Ab titers were determined by plaque reduction assay (20, 21).
Cytokines
For analysis of cytokine production, CLN cells from days 3, 5, and 7 days p.i. were cultured in 96-well plates (8 x 105 cells/well) in the presence or absence of JHMV Ag. Supernatants were harvested at 24 h for IL-2 and at 48 h for IL-4, IL-10, and IL-5 secretion. Cytokine concentrations were determined by ELISA and calculated from standard curves using commercially obtained recombinant cytokines as described previously (40). Detection limits were 75 pg/ml for IL-2, IL-5, and IL-10 and 125 pg/ml for IL-4.
RNA was isolated from brains by homogenization in guanidium isothiocyanate and centrifugation through cesium chloride as described previously (21, 40). cDNAs were prepared using avian myeloblastosis virus reverse transcriptase and oligo(dT) primers (Promega, Madison, WI) at 42°C. Cytokine mRNA was determined by semiquantitative PCR as described previously (20, 21, 40) using specific primer pairs for IL-5 (5' oligoprimer, 5'-GGT TAC AGA CAT GCA CCA TTG CCA CTA GTT-3'; 3' oligoprimer, 5'-CTA GTG GGT ATT AAA TTG AAG TTA GAT AGG-3') (41), IL-4, and IL-10 (21).
PCR products were quantitated by hybridization with [32P]ATP-labeled internal oligoprobes in a dot blot assay. Membranes were exposed toan imaging screen (Molecular Dynamics, Sunnyvale, CA) and analyzed using a PhosphorImager scanner (Molecular Dynamics). Cytokine cDNAs were normalized to the housekeeping gene HPRT to adjust for cDNAvariations (21, 40).
Cytolytic activity
CTL activity was measured using CLN cells obtained at 6 days
p.i. and spleen cells at various days p.i. (days 7, 9, and 14
p.i.) in a 51Cr release assay as described previously (16, 20). Cells were incubated for 7 days in the presence of the 1 µM S510
peptide (20, 42, 43). EL-4 (H-2b) target cells were coated
with 1 µM S510 peptide before use as targets (20, 42). Effectors were
added to the target cells at various E:T ratios and 51Cr
release measured after 4 h incubation at 37°C. Data are
expressed as specific 51Cr release. Spontaneous release
values were 
20% of the total release values.
Sequence analysis of Spike protein CTL epitope
Viruses from brain samples obtained at 21 days p.i. were
propagated once on confluent monolayers of DBT cells. When 
80% of
the cells exhibited cytopathology (24–48 h), cells were lysed by
addition of guanidine thyiocyanate solution and RNA isolated by
phenol/chloroform extraction. RNA prepared from DBT cells infected with
parental 2.2v-1 virus and uninfected DBT cells were used as controls
for mutations incorporated by Taq polymerase and for PCR
contamination respectively. RNA (5 µg) was reverse transcribed using
avian myeloblastosis virus reverse transcriptase and random
hexanucleotide primers (Promega). A 27-nucleotide cDNA encompassing the
S protein CTL epitope (viral bases 1528–1554) plus surrounding 500
bases (1420–1890) was amplified for 45 cycles using Amplitaq Gold
Polymerase (Perkin–Elmer, Branchburg, NJ) and primers JS 1895, 5'-GCA
TGC TAC GTT ATG TCC AGG CTG AGT C-3'; and JS 1390, 5'-GAT GTT
GCC TAC GCC CAG C-3'). Excess primers were removed using the Magic PCR
Prep (Promega), and the cDNA was sequenced on an ABI Prism automated
sequencing apparatus (Applied Biosciences, Foster City, CA) using JS
1390 as primer.
Histopathological analysis
Brain and spinal cord tissues were fixed in Clark’s solution for 3 h and prepared for paraffin sections as described previously (16, 20). Sections were stained with hematoxylin and eosin or Luxol fast blue for routine examination. To examine viral Ag distribution, sections were incubated with the anti-JHMV mAb J.3.3 specific for the nucleocapsid protein of JHMV (44) and immunoperoxidase-labeled anti-mouse mAb as secondary Ab (Vectastain-ABC kit, Vector Laboratories, Burlingame, CA). Multiple serial-step frozen sections of the brain tissue of 2 mice at day 14 p.i. were stained with mAb J.3.3 Ab. Ag-positive cells were counted to compare the number of infected cells in both study groups. Similarly, five serial-step paraffin sections of the spinal cord of three to four mice per group at day 21 p.i. were stained with Luxol fast blue, and the number of demyelination plaques was counted per unit area.
To examine CD4+ and CD8+ cells, immunoperoxidase staining was performed in acetone fixed frozen sections as described previously (45). In brief, rat anti-CD4 (L3T4, PharMingen, San Diego, CA) and rat anti-CD8 (Ly-2, PharMingen) were used as primary Ab. Visualization was achieved using biotinylated rabbit-anti-rat Ab, the Vectastain ABC kit and peroxidase substrate kit with aminoethylcarbizole (AEC) as chromogen (Vector Laboratories). For quantitative comparisons, five serial-step frozen sections of individual samples from groups of 3 mice at day 7 p.i. and 2 mice at day 14 p.i. were stained. All CD8+ T cells were counted.
To phenotype viral Ag-positive cells, sections were stained using the JHMV specific mAb J.3.3 and Ab specific for astrocytes (glial fibrillary acidic protein (GFAP; Dako, Carpinteria, CA), oligodendroglia (Rip; generously provided by Regeneron Pharmaceuticals, Tarrytown, NY (46)), or microglia (CD11b; PharMingen). JHMV Ag and GFAP double staining was performed on paraffin sections and visualized with peroxidase Vector VIP and Vector SG substrate kits (both from Vector Laboratories), respectively. Viral Ag in oligodendroglia was detected in frozen sections from paraformaldehyde-perfused tissue and detected using the alkaline phosphatase Vectastain ABC kit for Rip Ab and the peroxidase Vectastain ABC kit with AEC substrate for detecting J.3.3 mAb. Viral Ag in CD11b-positive cells was detected in acetone-fixed frozen sections, using alkaline phosphatase Vectastain ABC kit for CD11b and peroxidase Vectastain ABC kit with AEC substrate for J.3.3 mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0/0 mice
Morbidity and mortality were compared in IFN-0/0
and wt mice infected with JHMV until 21 days p.i. at which time
infected wt mice recovered from acute disease (Fig. 1
) (20, 21, 39). Animals in both groups
began to exhibit encephalitis at 7 days p.i., which progressed to a
paralytic disease (clinical score = 2.0 ± 0.2 for wt and
2.7 ± 0.2 for IFN-0/0 mice at 14 days p.i.) (Fig. 1A). Wt mice exhibited almost total clinical recovery by 21
days p.i. (clinical score = 1.2 ± 0.2). By contrast,
infected IFN-0/0 mice rapidly progressed to a paralytic
disease by 14 days p.i. and exhibited both slower clinical recovery and
higher mortality (clinical score = 2.6 ± 0.6 at 21 days
p.i.). Only 34% ± 8% of JHMV-infected IFN-0/0 mice
survived infection compared with 89% ± 8% of control mice indicating
that IFN- influences both survival and clinical course of
JHMV-induced encephalomyelitis (Fig. 1B).
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0/0 mice compared with controls at all time points
examined (Fig. 1
C). In contrast to wt mice, in which
infectious virus was no longer detected after day 7 p.i.,
infectious virus (
102 plaque forming units/g of brain)
persisted in the CNS of all IFN-0/0 mice until 21 days
p.i., the last time point examined (Fig. 1C). To rule out
reversion of the 2.2v-1 variant to wt JHMV, virus isolated from the
brains of IFN-0/0 mice at 21 days p.i. was assayed for
plaque formation in the presence or absence of mAb J.2.2 (used for
selection of JHMV neutralizing escape variant 2.2v-1) (39). No
differences in either plaque numbers or morphology was detected,
consistent with the absence of in vivo reversion (data not shown).
Immune responses in infected IFN-0/0 Mice
Infected IFN-0/0 mice were analyzed for
JHMV-specific CTL activity to determine whether the inability to clear
infectious virus was due to altered effector function. CTL activity in
CLN cells of JHMV-infected IFN-0/0 mice was identical to
the activity in wt mice during virus clearance from the CNS (Fig. 2), indicating that the absence of
IFN- did not diminish CTL activity. JHMV isolated during infection
of neonatal mice partially protected via maternal Ab, exhibit a high
mutation rate within the viral H-2b-restricted CTL epitope
allowing viral escape from CTL recognition (25, 26). To examine the
possibility that CTL escape mutants contributed to the persistence of
infectious virus within the CNS of IFN-0/0 mice, the S
protein mRNA encoding the CTL epitope (RNA bases 1528–1554) as well as
500 bases (1420–1890) surrounding the epitope was analyzed for
mutations. No dominant base substitutions or deletions within these S
protein sequences were detected by sequencing of bulk PCR products for
virus pools isolated from the CNS of persistently infected
IFN-0/0 mice at 21 days p.i. after infectious virus had
been completely eliminated from the CNS of wt mice. These data are
consistent with a direct antiviral effect of IFN- on JHMV
replication within the CNS.
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0/0 mice may reside in preferential induction of
Th2-type responses. No differences in proliferative responses to either
JHMV Ag or the CTL-specific S510 peptide were detected in either
splenocytes or CLN cells from infected IFN-0/0 mice
compared with wt mice at 7 or 14 days p.i. (data not shown). However,
CLN isolated from infected IFN-0/0 mice at 3 and 5 days
p.i. secreted more IL-2 in response to JHMV Ag compared with infected
wt controls (Fig. 3). IL-4 secretion was
not detected from cells derived from either group (data not shown).
IL-5 and IL-10 were secreted at higher levels from CLN derived from
infected IFN-0/0 mice at 7 days p.i. (Fig. 3). No
differences were detected in the quantity of IL-10 mRNA; however, less
IL-5 mRNA was detected in the CNS of infected IFN-0/0
mice compared with wt controls (Fig. 3). The diminished expression of
IL-5 mRNA and transient expression of IL-4 mRNA at day 7 p.i.
(Fig. 3) in IFN-0/0 mice suggest the absence of
increased trafficking or retention of Th2 cytokine secreting cells
within the CNS in the absence of IFN-. This contrasts with an
initial increase in Ag-specific secretion of Th2 type cytokines (IL-5
and IL-10) by peripheral T cells.
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0/0 mice compared with wt controls, whereas a
10-fold increase in IgG2a was initially detected in wt mice compared
with IFN-0/0 mice at day 9 p.i. (Fig. 4). Although the IFN-0/0
mice continued to have increased serum JHMV-specific IgG1 at 14 days
p.i., by 21 days p.i., the levels of both isotypes were equivalent in
the IFN-0/0 and wt mice (Fig. 4). As reported previously
(20, 21), no serum neutralizing Ab was detected before day 9 p.i.
and no differences were detected in JHMV neutralizing titers at 14 or
21 days p.i. (data not shown) suggesting that secretion of neutralizing
Ab was not altered in the absence of IFN-.
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Spinal cords and brains from infected IFN-0/0 mice
were compared with infected wt mice to determine the influence of the
absence of IFN- on viral Ag distribution and extent of pathological
changes. Prominent demyelination was found within white matter tracts
of both groups after 21 days p.i. Semiquantitative analysis showed no
significant differences in demyelination plaque numbers between
IFN-0/0 (2.4 ± 1.4) and wt (3.4 ± 2.l) mice
suggesting that IFN- does not influence development of JHMV-induced
demyelination. Similarly, no differences in the overall amount of
mononuclear inflammation were noted; however, an increase in the amount
of CD8+ T cells was detected in the white matter tracts of
infected IFN-0/0 mice at 7 and 14 days p.i. compared
with wt mice. Semiquantitative estimation of CD8+ T cells
infiltrating the brain parenchyma of infected mice showed a 4-fold
increase in the number of CD8+ cells in
IFN-0/0 mice at days 7 and 14 p.i. (200 ± 80
and 750 ± 210) compared with wt mice (30 ± 10 and 110
± 50). Interestingly, increased CD8+ infiltration in the
white matter regions correlated with increased Ag-positive cells in
IFN-0/0 mice suggesting that the lack of IFN- results
in increased viral Ag promoting local infiltration of CD8+
T cells.
Differences in the amount and type of Ag-positive cells were observed
beginning at day 7 p.i. (Fig. 5,
A and B). Decreasing numbers of viral Ag-positive
cells were observed in wt mice, consistent with the clearance of
infectious virus (Fig. 1C). By contrast, subsequent to day
7 p.i., significant Ag persisted until day 21 p.i. in
IFN-0/0 mice (Fig. 6,
A and B). By day 14 p.i. the
IFN-0/0 mice had a 10-fold increase in numbers of viral
Ag-positive cells (IFN-0/0 mice = 1500 ± 780
vs wt mice = 75 ± 60) compared with wt mice. Consistent with
the presence of infectious virus in infected IFN-0/0
mice, oligodendroglia, astrocytes, microglia, and occasional neurons
contained viral Ag; however, double staining for viral Ag in
astrocytes, microglia, and oligodendrocyte revealed that
oligodendroglia accounted for the majority of Ag-positive cells within
the CNS of IFN-0/0 mice at day 21 p.i. (Fig. 7A). These data are consistent
with the inability of CTL to mediate clearance from oligodendrocytes
(16, 20) and demonstrate that IFN- is indeed an important immune
effector in controlling JHMV infection of oligodendroglia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is
produced by both CD4+ and CD8+ T cells in
response to viral infections (49, 50). However, it is essential for
virus elimination during some (29, 30, 31, 32, 48) but not all (7, 51)
infections. Direct antiviral effects of IFN- appear to be not only
dependent upon the type of infection but also the tissue or cell type
infected. For example, IFN- contributes to viral clearance from the
periphery but not CNS (52), or from the CNS but not peripheral organs
(36). Although JHMV was partially cleared from the CNS of
IFN-0/0 mice, infectious virus persisted. Consistent
with the ability of the IFN-0/0 mice to exert partial
control of JHMV replication, CTL effectors were not inhibited in the
absence of IFN-. These data support previous observations that other
cytokines, including IL-2, IL-4, IL-6, and IL-10, in addition to
IFN- contribute to CTL induction (50). Thus, it appears that CTL
play a role in local viral inhibition even in the absence of IFN-
probably via direct perforin-mediated cytolysis of infected astrocytes
and microglia (16, 20). Infection of IFN-0/0 mice with a
JHMV variant whose replication is predominantly restricted to neurons
demonstrated that IFN- plays little or no role in the elimination of
JHMV from neurons (7), similar to the inability of CTL to mediate the
clearance of LCMV from neurons (12). By contrast, elimination of
measles virus from neurons is mediated by a CD4+ T cell
population (53), possibly due to the local secretion of IFN- (30).
The mechanism(s) of JHMV clearance from neurons is not clear; however,
the majority of JHMV is cleared from the CNS of IFN-0/0
mice, supporting the important role of CTL in regulating virus
replication in astrocytes and microglia (16).
Vector-mediated expression of IFN- from CNS cells infected with a
different strain of neurotropic coronavirus enhanced recruitment of
mononuclear cells and decreased virus replication (54). These data
suggested that IFN- acts at multiple levels by influencing cell
recruitment as well as serving as an antiviral effector. Although the
majority of virus was eliminated from the CNS of IFN-0/0
mice, infectious virus and increased numbers of Ag-positive cells
(mainly oligodendrocytes, but also a few glial cells and rare neurons)
remained in the CNS of IFN-0/0 mice in contrast to the
complete elimination of infectious virus and dramatic reduction in the
number of Ag-positive cells in wt mice. In the absence of IFN- no
differences in total infiltrating mononuclear cells were observed in
the brains of either neuronotropic OBLV-60 variant (7) or JHMV-infected
IFN-0/0 mice compared with wt controls. These data
suggest that IFN- similar to TNF- (55) is not an absolute
prerequisite for inflammatory cell entry into the CNS or the associated
loss of blood–brain barrier integrity during acute JHMV induced
encephalitis. The absence of IFN- resulted in increased
CD8+ T cell infiltration into white matter regions.
Increased CD8+ T cells correlated with increased
Ag-positive oligodendrocytes, suggesting that CTL accumulate within the
CNS even when they appear unable to effectively inhibit virus
replication within oligodendroglia. By contrast, no increased
CD8+ T cells were observed in IFN-0/0 mice
infected with a neuronotropic (OBLV-60) variant (7), suggesting
clearance from neurons via a IFN--independent mechanism. No
differences in the number of demyelinating plaques was observed in the
absence of IFN- suggesting that JHMV-induced lesion formation
requires additional inflammatory mechanisms. These data support the
notion that IFN- contributes to viral clearance but that it does not
directly influence macrophage-mediated demyelination. Nevertheless it
is possible that the equivalent demyelination in JHMV-infected
IFN-0/0 mice occurs by an effector mechanism that
differs from autoimmune demyelination (56, 57) or alternatively it may
be due to direct effects of JHMV replication in oligodendroglia (8)
associated with necrotic, rather than apoptotic cell death (20).
The immune-mediated encephalomyelitis induced by JHMV infection is
comprised of NK cells, virus specific CTL and CD4+ T cells
and monocytes. The emerging picture of this complex infection that
results in viral persistence and chronic demyelination is that specific
immune effector mechanisms contribute to the control of virus
replication within specific subsets of the major CNS cells types.
Although NK cells are rapidly recruited, they appear to play little or
no role in this infectious process (6, 23). Similarly, neutralizing Ab
is detected only after the majority of virus is cleared (20, 21),
suggesting that it plays little or no role in limiting virus
replication. By contrast, the virus-specific CTL response appears to
limit acute infection of both astrocytes and microglia (16).
JHMV-specific induction of CTL was initially reported to be dependent
on CD4+ T cells (22); however, recent data demonstrate that
CTL are induced and traffic normally into the CNS during infection of
CD4-depleted hosts (45). The inability to limit virus in the absence of
CD4+ T cells is related to a requirement for the
maintenance of CTL viability within the CNS. Therefore,
CD4+ T cells play a critical role in CTL effector function
within the CNS, in addition to their potential direct role in virus
clearance (15, 53). This report demonstrates that IFN- contributes
to the overall inhibition of virus replication specifically in
oligodendroglia. Thus, both virus-specific cytolytic activity and
IFN- appear to differentially inhibit JHMV replication within the
CNS by exhibiting cell type specific effector function. The inability
of CTL to inhibit replication in oligodendroglia may reflect the
absence of MHC class I expression, similar to the inability of CTL
specific for LCMV to limit virus replication in neurons (12).
Expression of the IFN- receptor on oligodendroglia (37) and the
recent suggestion that IFN- may be critical in limiting measles
virus expression in neurons (30) both support a vital role for this
cytokine in limiting virus infections within specific CNS cell types.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen Stohlman, University of Southern California School of Medicine, 1333 San Pablo Street, MCH 142, Los Angeles, CA 90033. E-mail address:
3 Abbreviations used in this paper: CNS, central nervous system; CLN, cervical lymphoid nodes; IFN-0/0, homozygous IFN- knockout mice; JHMV, JHM strain of mouse hepatitis virus; LCMV, lymphocytic choriomengitis virus; wt, wild type; p.i., postinfection; GFAP, glial fibrillary acidic proteins; AEC, aminoethylcarbizole.
Received for publication May 29, 1998. Accepted for publication October 13, 1998.
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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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K. C. MacNamara, S. J. Bender, M. M. Chua, R. Watson, and S. R. Weiss Priming of CD8+ T Cells during Central Nervous System Infection with a Murine Coronavirus Is Strain Dependent J. Virol., July 1, 2008; 82(13): 6150 - 6160. [Abstract] [Full Text] [PDF]
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K. B. Walsh, M. B. Lodoen, R. A. Edwards, L. L. Lanier, and T. E. Lane Evidence for Differential Roles for NKG2D Receptor Signaling in Innate Host Defense against Coronavirus-Induced Neurological and Liver Disease J. Virol., March 15, 2008; 82(6): 3021 - 3030. [Abstract] [Full Text] [PDF]
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K. B. Walsh, L. L. Lanier, and T. E. Lane NKG2D Receptor Signaling Enhances Cytolytic Activity by Virus-Specific CD8+ T Cells: Evidence for a Protective Role in Virus-Induced Encephalitis J. Virol., March 15, 2008; 82(6): 3031 - 3044. [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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T. A. Miura, E. A. Travanty, L. Oko, H. Bielefeldt-Ohmann, S. R. Weiss, N. Beauchemin, and K. V. Holmes The Spike Glycoprotein of Murine Coronavirus MHV-JHM Mediates Receptor-Independent Infection and Spread in the Central Nervous Systems of Ceacam1a / Mice J. Virol., January 15, 2008; 82(2): 755 - 763. [Abstract] [Full Text] [PDF]
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K. B. Walsh, R. A. Edwards, K. M. Romero, M. V. Kotlajich, S. A. Stohlman, and T. E. Lane Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease J. Immunol., July 15, 2007; 179(2): 1155 - 1165. [Abstract] [Full Text] [PDF]
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P. Banerjee, R. Rochford, J. Antel, G. Canute, S. Wrzesinski, M. Sieburg, and G. Feuer Proinflammatory Cytokine Gene Induction by Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2 Tax in Primary Human Glial Cells J. Virol., February 15, 2007; 81(4): 1690 - 1700. [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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 Y. Wang, M. Lobigs, E. Lee, A. Koskinen, and A. Mullbacher CD8+ T cell-mediated immune responses in West Nile virus (Sarafend strain) encephalitis are independent of gamma interferon J. Gen. Virol., December 1, 2006; 87(12): 3599 - 3609. [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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 S. R. Weiss and S. Navas-Martin Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus Microbiol. Mol. Biol. Rev., December 1, 2005; 69(4): 635 - 664. [Abstract] [Full Text] [PDF]
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J. Hausmann, A. Pagenstecher, K. Baur, K. Richter, H.-J. Rziha, and P. Staeheli CD8 T Cells Require Gamma Interferon To Clear Borna Disease Virus from the Brain and Prevent Immune System-Mediated Neuronal Damage J. Virol., November 1, 2005; 79(21): 13509 - 13518. [Abstract] [Full Text] [PDF]
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K. Obojes, O. Andres, K. S. Kim, W. Daubener, and J. Schneider-Schaulies Indoleamine 2,3-Dioxygenase Mediates Cell Type-Specific Anti-Measles Virus Activity of Gamma Interferon J. Virol., June 15, 2005; 79(12): 7768 - 7776. [Abstract] [Full Text] [PDF]
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R. Wahid, M. J. Cannon, and M. Chow Virus-Specific CD4+ and CD8+ Cytotoxic T-Cell Responses and Long-Term T-Cell Memory in Individuals Vaccinated against Polio J. Virol., May 15, 2005; 79(10): 5988 - 5995. [Abstract] [Full Text] [PDF]
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K. Nakagaki, K. Nakagaki, and F. Taguchi Receptor-Independent Spread of a Highly Neurotropic Murine Coronavirus JHMV Strain from Initially Infected Microglial Cells in Mixed Neural Cultures J. Virol., May 15, 2005; 79(10): 6102 - 6110. [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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C. Ramakrishna, S. A. Stohlman, R. A. Atkinson, D. R. Hinton, and C. C. Bergmann Differential Regulation of Primary and Secondary CD8+ T Cells in the Central Nervous System J. Immunol., November 15, 2004; 173(10): 6265 - 6273. [Abstract] [Full Text] [PDF]
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M. Suresh, X. Gao, C. Fischer, N. E. Miller, and K. Tewari Dissection of Antiviral and Immune Regulatory Functions of Tumor Necrosis Factor Receptors in a Chronic Lymphocytic Choriomeningitis Virus Infection J. Virol., April 15, 2004; 78(8): 3906 - 3918. [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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M. M. Chua, K. C. MacNamara, L. San Mateo, H. Shen, and S. R. Weiss Effects of an Epitope-Specific CD8+ T-Cell Response on Murine Coronavirus Central Nervous System Disease: Protection from Virus Replication and Antigen Spread and Selection of Epitope Escape Mutants J. Virol., February 1, 2004; 78(3): 1150 - 1159. [Abstract] [Full Text] [PDF]
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M. J. Trifilo, C. Montalto-Morrison, L. N. Stiles, K. R. Hurst, J. L. Hardison, J. E. Manning, P. S. Masters, and T. E. Lane CXC Chemokine Ligand 10 Controls Viral Infection in the Central Nervous System: Evidence for a Role in Innate Immune Response through Recruitment and Activation of Natural Killer Cells J. Virol., January 15, 2004; 78(2): 585 - 594. [Abstract] [Full Text] [PDF]
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M. Rodriguez, L. J. Zoecklein, C. L. Howe, K. D. Pavelko, J. D. Gamez, S. Nakane, and L. M. Papke Gamma Interferon Is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection J. Virol., November 15, 2003; 77(22): 12252 - 12265. [Abstract] [Full Text] [PDF]
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 J. J. Hooks, Y. Wang, and B. Detrick The Critical Role of IFN-{gamma} in Experimental Coronavirus Retinopathy Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3402 - 3408. [Abstract] [Full Text] [PDF]
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C. Ramakrishna, C. C. Bergmann, R. Atkinson, and S. A. Stohlman Control of Central Nervous System Viral Persistence by Neutralizing Antibody J. Virol., April 15, 2003; 77(8): 4670 - 4678. [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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N. W. Marten, S. A. Stohlman, J. Zhou, and C. C. Bergmann Kinetics of Virus-Specific CD8+-T-Cell Expansion and Trafficking following Central Nervous System Infection J. Virol., February 15, 2003; 77(4): 2775 - 2778. [Abstract] [Full Text] [PDF]
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 R. G. v. d. Most, K. Murali-Krishna, and R. Ahmed Prolonged presence of effector-memory CD8 T cells in the central nervous system after dengue virus encephalitis Int. Immunol., January 1, 2003; 15(1): 119 - 125. [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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K. Kimura, K. Kakimi, S. Wieland, L. G. Guidotti, and F. V. Chisari Activated Intrahepatic Antigen-Presenting Cells Inhibit Hepatitis B Virus Replication in the Liver of Transgenic Mice J. Immunol., November 1, 2002; 169(9): 5188 - 5195. [Abstract] [Full Text] [PDF]
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D. J. J. Carr and S. Noisakran The Antiviral Efficacy of the Murine Alpha-1 Interferon Transgene against Ocular Herpes Simplex Virus Type 1 Requires the Presence of CD4+, {alpha}/{beta} T-Cell Receptor-Positive T Lymphocytes with the Capacity To Produce Gamma Interferon J. Virol., August 12, 2002; 76(18): 9398 - 9406. [Abstract] [Full Text] [PDF]
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P. T. Massa, S. L. Ropka, S. Saha, K. L. Fecenko, and K. L. Beuler Critical Role for Protein Tyrosine Phosphatase SHP-1 in Controlling Infection of Central Nervous System Glia and Demyelination by Theiler's Murine Encephalomyelitis Virus J. Virol., July 17, 2002; 76(16): 8335 - 8346. [Abstract] [Full Text] [PDF]
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J. Zhou, S. A. Stohlman, R. Atkinson, D. R. Hinton, and N. W. Marten Matrix Metalloproteinase Expression Correlates with Virulence following Neurotropic Mouse Hepatitis Virus Infection J. Virol., June 27, 2002; 76(15): 7374 - 7384. [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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C. E. Patterson, D. M. P. Lawrence, L. A. Echols, and G. F. Rall Immune-Mediated Protection from Measles Virus-Induced Central Nervous System Disease Is Noncytolytic and Gamma Interferon Dependent J. Virol., March 27, 2002; 76(9): 4497 - 4506. [Abstract] [Full Text] [PDF]
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M. S. Ibrahim, M. Watanabe, J. A. Palacios, W. Kamitani, S. Komoto, T. Kobayashi, K. Tomonaga, and K. Ikuta Varied Persistent Life Cycles of Borna Disease Virus in a Human Oligodendroglioma Cell Line J. Virol., March 19, 2002; 76(8): 3873 - 3880. [Abstract] [Full Text] [PDF]
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S.-I. Tschen, C. C. Bergmann, C. Ramakrishna, S. Morales, R. Atkinson, and S. A. Stohlman Recruitment Kinetics and Composition of Antibody-Secreting Cells Within the Central Nervous System Following Viral Encephalomyelitis J. Immunol., March 15, 2002; 168(6): 2922 - 2929. [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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C. Ramakrishna, S. A. Stohlman, R. D. Atkinson, M. J. Shlomchik, and C. C. Bergmann Mechanisms of Central Nervous System Viral Persistence: the Critical Role of Antibody and B Cells J. Immunol., February 1, 2002; 168(3): 1204 - 1211. [Abstract] [Full Text] [PDF]
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J. Hausmann, K. Schamel, and P. Staeheli CD8+ T Lymphocytes Mediate Borna Disease Virus-Induced Immunopathology Independently of Perforin J. Virol., November 1, 2001; 75(21): 10460 - 10466. [Abstract] [Full Text] [PDF]
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A. E. Matthews, S. R. Weiss, M. J. Shlomchik, L. G. Hannum, J. L. Gombold, and Y. Paterson Antibody Is Required for Clearance of Infectious Murine Hepatitis Virus A59 from the Central Nervous System, But Not the Liver J. Immunol., November 1, 2001; 167(9): 5254 - 5263. [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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C. C. Bergmann, C. Ramakrishna, M. Kornacki, and S. A. Stohlman Impaired T Cell Immunity in B Cell-Deficient Mice Following Viral Central Nervous System Infection J. Immunol., August 1, 2001; 167(3): 1575 - 1583. [Abstract] [Full Text] [PDF]
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M. T. Liu, D. Armstrong, T. A. Hamilton, and T. E. Lane Expression of Mig (Monokine Induced by Interferon-{{gamma}}) Is Important in T Lymphocyte Recruitment and Host Defense Following Viral Infection of the Central Nervous System J. Immunol., February 1, 2001; 166(3): 1790 - 1795. [Abstract] [Full Text] [PDF]
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M. T. Liu, B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, and T. E. Lane Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease J. Immunol., September 1, 2000; 165(5): 2327 - 2330. [Abstract] [Full Text] [PDF]
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G. F. Wu, L. Pewe, and S. Perlman Coronavirus-Induced Demyelination Occurs in the Absence of Inducible Nitric Oxide Synthase J. Virol., August 15, 2000; 74(16): 7683 - 7686. [Abstract] [Full Text]
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G. F. Wu, A. A. Dandekar, L. Pewe, and S. Perlman CD4 and CD8 T Cells Have Redundant But Not Identical Roles in Virus-Induced Demyelination J. Immunol., August 15, 2000; 165(4): 2278 - 2286. [Abstract] [Full Text] [PDF]
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N. W. Marten, S. A. Stohlman, R. D. Atkinson, D. R. Hinton, J. O. Fleming, and C. C. Bergmann Contributions of CD8+ T Cells and Viral Spread to Demyelinating Disease J. Immunol., April 15, 2000; 164(8): 4080 - 4088. [Abstract] [Full Text] [PDF]
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B. Parra, M. T. Lin, S. A. Stohlman, C. C. Bergmann, R. Atkinson, and D. R. Hinton Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System J. Virol., March 1, 2000; 74(5): 2447 - 2450. [Abstract] [Full Text]
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G. F. Wu and S. Perlman Macrophage Infiltration, but Not Apoptosis, Is Correlated with Immune-Mediated Demyelination following Murine Infection with a Neurotropic Coronavirus J. Virol., October 1, 1999; 73(10): 8771 - 8780. [Abstract] [Full Text] [PDF]
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C. C. Bergmann, J. D. Altman, D. Hinton, and S. A. Stohlman Inverted Immunodominance and Impaired Cytolytic Function of CD8+ T Cells During Viral Persistence in the Central Nervous System J. Immunol., September 15, 1999; 163(6): 3379 - 3387. [Abstract] [Full Text] [PDF]
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