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Departments of
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Pathology,
Neurology, and
Molecular Microbiology and Immunology, 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(6, 7, 8).
However, the immune response to JHMV infection appears to be only
partially effective. Although infectious virus, and the accompanying
immunopathology, is eliminated from the CNS, JHMV persists in a
noninfectious form associated with chronic ongoing demyelination, which
pathologically resembles the chronic human demyelinating disease,
multiple sclerosis. The role(s) of anti-viral effectors during acute self-limiting viral infections is becoming increasingly clear; however, their role(s) in establishing or maintaining persistent viral infections is less well understood. For example, clearance of acute LCMV infection is largely dependent upon the CTL response (2). However, long-term control of LCMV is complex and appears to be dependent upon humoral immunity, CD4+ T cells, and cytokines (9, 10). Similarly, anti-viral Ab responses, influenced by CD4+ T cells, promote recovery from acute influenza virus infection of the murine respiratory tract (11), while virus-specific CTL are the major anti-viral effectors (12). These data suggest that, in addition to a role in controlling some cytopathic viruses, humoral immunity may be a necessary effector mechanism required to control persistent viral infections (13, 14).
A common outcome of viral infection of the CNS is persistence. The presence of the blood-brain barrier, restricted expression of MHC molecules, and the nonrenewable nature of neurons are all unique characteristics that may hinder viral clearance and help establish persistent CNS viral infections (15). For viruses to persist, they must assume a nonlytic phenotype and/or alter gene expression to avoid immune surveillance. For example, noncytopathic viruses may circumvent CTL recognition by infection of MHC class I-negative neurons (16) or by suppressing virus-specific T cell responses during CNS persistence (17). However, Sindbis virus infection of neurons is controlled via an Ab-mediated mechanism distinct from Ab-dependent cell-mediated cytotoxicity or C-dependent lysis (14, 18). In contrast to protection from cytopathic viruses provided by Ab, the virus-specific Ab response modulates viral gene expression and helps maintain persistence following measles virus (19, 20), Sindbis virus (21), and herpes simplex virus infections (22). Viruses with high mutation rates, especially RNA viruses such as JHMV, may persist in the CNS by generating attenuated variants or altering epitopes in response to immune pressure (23, 24).
Infection of IgM-/- mice with JHMV was examined to determine whether the humoral response influenced clearance during acute infection or the ability to establish a persistent CNS infection. The passive transfer of both neutralizing and, in some instances, nonneutralizing mAb before JHMV infection provides protection from lethal disease (25, 26, 27, 28, 29). However, during acute infection JHMV is cleared from the CNS before the detection of serum-neutralizing Ab responses (6). Consistent with data suggesting that cell-mediated immunity is the primary effector of JHMV clearance, the absence of a humoral response did not influence initial clearance of virus from the CNS. However, following initial clearance, reactivation of infectious virus was found in the CNS of IgM-/-, but not control mice. The passive transfer of anti-viral Ab after the majority of infectious virus was cleared from the CNS, but before virus reactivation, prevented the reappearance of infectious virus and diminished immunopathology. These data demonstrate that in spite of an effective cell-mediated immune response initially able to control infectious virus, humoral immunity can maintain control of infectious virus within the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of C57BL/6-Igh-6tm1Cgn (IgM-/-) mice (30) and syngeneic C57BL/6 (wt) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Homozygous IgM-/- mice were bred at the University of Southern California School of Medicine (Los Angeles, CA) under pathogen-free conditions. The absence of Ig in IgM-/- mice was confirmed by testing sera for murine IgG by ELISA, as previously described (31). All mice used were between the ages of 7–8 wk of age.
Viral infection and titer determination
Mice were infected via intracerebral inoculation with 50 plaque-forming units of the 2.2v-1 mAb-derived variant of JHMV in 32 µl of Dulbecco’s PBS, pH 7.4, as previously described (32). The 2.2v-1 strain of JHMV was kindly supplied by J. Fleming (University of Wisconsin, Madison, WI). Tissue levels of infectious virus were determined by plaque assay on monolayers of DBT cells, as previously described (6). Briefly, clarified homogenates from one-half of the brains were serially diluted in serum-free-PBS containing 10% tryptose phosphate broth, and virus was adsorbed for 90 min at room temperature. Plaque numbers were determined following 24–48-h incubation at 37°C. Data represent the average of triplicate samples for groups of four or more mice.
Clinical disease
Clinical disease was graded as previously described (6, 28): 0, healthy; 1, ruffled fur and hunch back; 2, slow mobility and inability to upright; 3, paralysis and wasting; 4, moribund and death. Data represent the average for four or more mice per group.
CTL assays
Spleen cell suspensions were prepared at 7, 11, and 21 days
postinfection (p.i.). Splenocytes from three mice per group were
pooled, and 1 x 108 cells were cultured for 6 days at
37°C with irradiated (25 Gy) syngeneic JHMV-infected spleen cells in
RPMI 1640 medium supplemented with 10% FCS (Gemini Biological
Products, Calabasas, CA), 2 mM glutamine, 25 µg/ml gentamicin, 1 mM
sodium pyruvate, 5 x 10-5 M 2-ME, and nonessential
amino acids. Cytolytic activity was measured as previously described
(6, 7). Briefly, syngeneic EL-4 (H-2b) target cells were
infected with either a recombinant vaccinia virus (rVV) expressing
amino acids 510–518 of the JHMV spike (S) protein (vJS510) or a
control rVV expressing the Escherichia coli lacZ gene (vSC8)
at a multiplicity of infection of 5 (33). Following incubation for
14 h at 37°C, target cells were labeled with 100 µCi
Na51CrO4 (New England Nuclear, Boston, MA) for
1 h at 37°C. Effector cells were added to target cells at
various E:T ratios. 51Cr release was measured after 4-h
incubation at 37°C. Data are expressed as percent specific release
defined as: ((experimental release) - (spontaneous release))/((total
release - (spontaneous release)). Maximum spontaneous release
values were 
20% of total release.
RNA expression
Total RNA was prepared from one-half of spinal cords divided
longitudinally by homogenization in guanidine isothiocyanate and
isolated by centrifugation through 5.4 M cesium chloride at
100,000 x g for 18 h, as previously described
(31). The cDNA were prepared using avian myeloblastosis
reverse-transcriptase (Promega, Madison, WI) and oligo(dT) primers
(Promega) for 1 h at 42°C. PCR was performed using AmpliTaq DNA
polymerase (Perkin-Elmer, Branchburg, NJ) and primers specific for
IFN- (31) and JHMV nucleocapsid protein (N) (viral nucleotides
527-1257): 5'-ATA GGA TCC ATG GCT ACT AGG TTT GCG CCC GGC-3' and 5'-ACA
GTT ACC TAC ATC TGC ACC ACC ATC TTG-3'. Amplification of IFN- mRNA
was carried out using 35 cycles of one denaturing step for 45 s at
94°C, primer annealing for 45 s at 59°C, and extension for 1.5
min at 72°C. Amplification for JHMV N mRNA was conducted using 25
cycles of one denaturing step at 94°C (45 s), primer annealing at
72°C (45 s), extension step at 72°C (2 min), followed by a final
extension step for 7 min. For quantification, PCR products were diluted
in denaturing solution (0.4 N NaOH, 25 mM EDTA), neutralized with
Tris-HCl (1 M; pH 8), and transferred to 0.45-µm Nytran membranes
(Schleicher & Schuell, Keene, NH) using a Minifold I dot-blot
apparatus. Membranes were hybridized overnight at 60°C with
[32P]ATP-labeled internal oligonucleotide probes for
IFN- (31) and JHMV N mRNA: 5'-ATA GGA TCC ATG GTT TTG GCT AAG CTC
GGT AAA G-3'. Membranes were washed, exposed to Storage Phosphor
Screens (Molecular Dynamics, Sunnyvale, CA), and scanned using a
PhosphorImaging Scanner (Molecular Dynamics). To adjust for differences
in cDNA quantity, levels were normalized to the housekeeping enzyme
hypoxanthine phosphoriboxyltransferase, as previously described (31).
Mean values for three mice per group are presented.
Sequence analysis
Virus derived from brain samples was propagated once on DBT monolayers, and the cells were lysed by addition of guanidine thiocyanate solution when approximately 80% of the cells exhibited cytopathology. RNA was isolated by phenol/chloroform extraction. RNA from cells infected with parental virus and uninfected cells were used to control for mutations introduced by Taq polymerase and PCR contamination, respectively. RNA (5 µg) was reverse transcribed using avian myeloblastosis virus reverse-transcriptase and random hexanucleotide primers (Promega). A cDNA encompassing the immunodominant CTL epitope within the JHMV S protein (viral nucleotides 1528–1554) plus surrounding 500 nucleotides 1420–1890(1420–1890) was amplified using oligonucleotide primers JS1895 and JS1390, as previously described (8). The cDNAs were sequenced on an ABI Prism automated sequencing apparatus using oligonucleotide JS1390 as primer, as previously described (8).
Ab preparations
Serum-free supernatants from a hybridoma producing JHMV-neutralizing mAb J.2.2 (IgG2b) (34) were concentrated by precipitation with saturated ammonium sulfate. Anti-JHMV polyclonal Ab was obtained from mice hyperimmunized with JHMV (35). Anti-JHMV and control ascites were induced in immunized and naive mice, respectively, by injection of Sarcoma 180 cells (35). Ascites were defibrinated (36) and heat inactivated at 56°C for 30 min before use. Control ascites was tested by ELISA to insure the absence of JHMV Ab, as previously described (31).
Passive transfers
IgM-/- mice received 150 µg of either JHMV-neutralizing mAb J.2.2 or an equal amount of IgG2b myeloma protein (Zymed Laboratories, San Francisco, CA). These Ab and the polyclonal anti-JHMV and control ascites were transferred into the peritoneal cavity of infected IgM-/- mice at 9, 12, and 17 days p.i. Neither the isotype control myeloma protein nor the ascites obtained from naive mice had detectable anti-JHMV Ab, as determined by ELISA. Neutralization titers were determined by the plaque-reduction method (6, 31, 35). Briefly, 50–100 plaque-forming units of either DM (for mAb J.2.2) or the 2.2v-1 (for immune ascites) JHMV strains were mixed with serial 4-fold dilutions. After 1 h at 37°C, residual virus was determined by plaque assay. The dilution that reduced plaque numbers by 50% was designated as titer (35). The neutralizing titers of the J.2.2 mAb and anti-JHMV ascites were 1/1600 and 1/3200, respectively.
Histopathology
Brains, bisected in the mid-coronal plane, and spinal cords were prepared for frozen sections or embedded in paraffin. For paraffin preparations, tissues were fixed for 3 h in Clark’s solution (75% ethanol, 25% glacial acetic acid) before embedding. Sections were stained with either hematoxylin and eosin or luxol fast blue. Distribution of JHMV Ag was determined by immunoperoxidase staining (Vectastain-ABC kit; Vector Laboratories, Burlingame, CA) using the anti-JHMV mAb J.3.3 specific for the carboxyl terminus of the N protein as the primary Ab (34, 37) and horse anti-mouse mAb as secondary Ab (Vector Laboratories). CD4+ and CD8+ T cells were identified in frozen sections fixed with acetone for 2 min at room temperature and stained using anti-CD4 (L3T4; PharMingen, San Diego, CA) or anti-CD8 (Ly-2, PharMingen) mAb. Both primary Ab were detected with a biotinylated rabbit anti-rat serum preabsorbed with mouse serum (Vector Laboratories) and visualized using Vectastain-ABC kits. Apoptotic cells were identified using Oncor ApopTag Kit (Gaithersburg, MD), which utilizes TdT for extension of fragmented DNA, as previously described (6). Tissue processing and staining were performed according to the manufacturer’s instructions. For quantitative comparisons, four serial step sections of individual samples from groups of three to four mice were stained, and all positive cells counted. Student’s t test was used for statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether humoral immunity contributes to JHMV
pathogenesis, clinical disease and mortality were compared in infected
IgM-/- and wt mice. Both wt and IgM-/- mice
developed clinical signs of disease (mild ruffled fur and hunched back)
by 7–8 days p.i. (Fig. 1
A),
which progressed to hind limb paralysis and wasting in both groups by
approximately 13 days p.i. In contrast to the clinical recovery of wt
mice, IgM-/- mice showed signs of increasing lethargy and
wasting between 14 and 40 days p.i. (Fig. 1A). The increase
in clinical signs coincided with an increase in mortality of
IgM-/- mice after 25 days p.i. (Fig. 1B). By
day 40 p.i., only 8% of JHMV-infected IgM-/- mice
survived and the remainder appeared moribund. By contrast, 80% of wt
mice had completely recovered from infection and minor gait
abnormalities.
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). However, by 14 days p.i., when no
infectious JHMV could be isolated from the CNS of wt mice, infectious
virus had reemerged in the IgM-/- mice after 11 days p.i.
Between 14 and 35 days p.i., infectious JHMV continued to increase in
the CNS of the surviving IgM-/- mice. By contrast,
infectious virus remained undetectable in the CNS of the wt mice. These
data confirmed the role of cellular immunity in JHMV clearance during
acute CNS infection (6, 7) and suggest that the anti-viral Ab
response is critical for maintaining the suppression of infectious
virus, resulting in the establishment of a persistent CNS infection.
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To determine whether IgM-/- mice exhibited altered
inflammatory responses, the extent of mononuclear cell infiltration and
frequency of apoptotic cells in the CNS was compared with wt mice. At
day 7 p.i., the majority of the mononuclear cell infiltration and
apoptotic cells were localized to the perivascular areas, with only a
few CD4+ or CD8+ T cells detected within the
parenchyma in both groups (data not shown). At day 21 p.i., the
CNS of the IgM-/- mice showed increased numbers of
perivascular mononuclear cells (Fig. 3).
This distribution of inflammatory cells is reminiscent of the
perivascular inflammation observed during acute JHMV infection (38).
Indeed, a 10-fold increase in perivascular CD4+ T cells was
observed in the IgM-/- mice compared with the wt mice at
day 21 p.i. (Fig. 3). In both groups, the CD8+ T cells
were present in a diffuse pattern within the parenchyma (Fig. 3).
Although the numbers of CD8+ T cells varied dramatically in
individual microscopic fields, in contrast to the CD4+ T
cells, the total number of CD8+ T cells in the
IgM-/- mice was not statistically different from wt mice.
Fewer apoptotic cells were detected at 21 days p.i. compared with 7
days p.i.; however, no difference in the numbers or distribution of
apoptotic cells was observed comparing the two groups at 21 days p.i.
(Fig. 3). These data suggest, in contrast to the absence of
CD4+ T cells that dramatically increased the number of
apoptotic lymphocytes (38), the absence of humoral immunity did not
result in an increase in apoptosis of effector cells within the CNS.
Both wt and IgM-/- mice had prominent demyelination
within the spinal cord (Fig. 4). In
contrast to wt mice, demyelinating lesions were detected in the brains
of IgM-/- mice in areas in which virus Ag was found (data
not shown). These data suggest that the cell-mediated immune response
is not compromised in the IgM-/- mice, although the
increase in infectious virus is associated with increased
demyelination.
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IgM-/- mice and wt controls were infected with the
JHMV 2.2v-1 variant derived by selection from a highly virulent JHMV
with neutralizing mAb J.2.2 (32). A possible explanation for the
recrudescence of JHMV within the CNS of the IgM-/- mice
is reversion of the 2.2v-1 phenotype to a more virulent phenotype (39).
To examine this possibility, mAb J.2.2 was passively administered to
infected IgM-/- mice at 9, 12, and 17 days p.i. and
compared with infected IgM-/- mice receiving an
isotype-matched Ab with no detectable ability to recognize JHMV. These
time points were chosen to parallel the initial detection of
neutralizing Ab in JHMV-infected mice (6). Neither passive transfer of
mAb J.2.2 nor the control Ab altered the clinical course of infection
or prevented recrudescence of JHMV at 21 days p.i. (Table I). Furthermore, no difference in the
extent of inflammation or distribution of viral Ag was detected
comparing the mAb J.2.2 recipients, the control Ab recipients, or
untreated IgM-/- mice (data not shown). These data
suggest that reversion to the parental phenotype was not responsible
for the reactivation of JHMV in the CNS of the IgM-/-
mice following initial clearance.
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). Only rarely were neurons positive for
viral Ag in either group. At 11 days p.i., few Ag-positive cells were
detected in the brains of either group (Fig. 5), consistent with the
reduction in infectious virus (Fig. 2
). A similar cellular distribution
of viral Ag in both groups during the acute phase of disease (7–11
days p.i.) suggests that altered cellular tropism did not contribute to
the recrudescence of infectious JHMV in the absence of Ab. Similarly,
only focal areas containing viral Ag-positive cells were detected
within the spinal cords of wt mice at 21 days p.i. (Fig. 5), consistent
with the inability to recover infectious virus at this time point (Fig. 2). By contrast, the brains of IgM-/- mice showed
numerous Ag-positive cells (Fig. 5), consistent with the presence of
infectious virus (Fig. 2), and the spinal cords showed a more diffuse
distribution of Ag-positive cells. In both sites, virus Ag was
predominantly found in the white matter; however, focal involvement of
gray matter was also seen. The cellular distribution of virus Ag at 21
days p.i. in the IgM-/- mice was similar to that seen at
earlier time points with predominant localization to astrocytes,
oligodendrocytes, and microglia. A small number of Ag-positive neurons
was also present. With the exception of viral spread to a small number
of neurons, the distribution of viral Ag was comparable in the two
groups, suggesting no change in cell tropism in the absence of humoral
immunity.
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CTL responses in the spleens of IgM-/- and wt mice
were comparable at day 7 p.i. (Fig. 6), consistent with the predominant role
of these effectors in JHMV clearance from the CNS (6, 7). CTL activity
in the spleens of wt mice was equivalent to the day 7 p.i. level
at 21 days p.i. By contrast, peripheral CTL activity in
IgM-/- mice was reduced compared with the levels detected
in wt mice (Fig. 6), suggesting the retention of CTL within the CNS due
to the presence of infectious virus and viral Ag within the CNS (Figs. 2 and 5). Accumulation of IFN- mRNA and the mRNA encoding the N
protein of JHMV within the CNS of infected IgM-/- mice
was examined to demonstrate that the decreased peripheral CTL activity
in IgM-/- mice represented continuous recruitment and/or
sequestration in the CNS due to the presence of infectious virus (40).
The IFN- mRNA levels in the CNS of IgM-/- and wt mice
were approximately equivalent during acute infection and viral
clearance (7, 9, and 11 days p.i.; Fig. 7). The kinetics of accumulation of JHMV
N mRNA paralleled the kinetics of virus replication within the CNS of
both groups (Fig. 7). However, while the levels of IFN- mRNA in wt
mice decreased to below detection limits as infectious virus was
eliminated (Fig. 5), IFN- mRNA levels in the CNS of the
IgM-/- mice remained elevated (Fig. 7). Although both
CD4+ and CD8+ T cells could contribute to
IFN- mRNA within the CNS, CD8+ T cells are a major
source of this cytokine (41, 42), suggesting that reduced splenic CTL
in the IgM-/- mice is due to the presence of activated
CTL within the CNS (Fig. 3).
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T at nucleotide 1539), resulting in a WC change at
amino acid 513. None of the six cloned PCR products from another 30 day
p.i. mouse had any detectable mutations within the S gene. To determine
whether the WC mutation altered CTL recognition, viruses with both
the wt and mutant sequences were plaque purified from mice 30 days
p.i., and the presence of wt and mutation was confirmed by sequence
analysis. Target cells (IC-21 (H-2b)) infected with both
viruses were recognized by JHMV-specific CTL (data not shown). These
data suggest that mutations within the immunodominant CTL epitope can
indeed be obtained in the absence of humoral immunity. However, the
paucity of mutants obtained only well after virus reactivation supports
a functional cell-mediated immune response within the CNS of infected
IgM-/- mice. Passive transfer eliminates recrudescence of infectious JHMV
Serum JHMV-neutralizing Ab is detected approximately 9 days p.i.
in JHMV-infected wt mice (6). These data suggest that the Ab response
contributes to preventing JHMV recrudescence. To demonstrate that the
absence of an Ab response is responsible for the reactivation of
infectious virus, anti-JHMV Ab was passively transferred to
infected IgM-/- mice at 9, 12, and 17 days p.i., and its
ability to inhibit CNS virus reactivation was determined at 21 days
p.i. Control IgM-/- mice received an equal volume of
ascites obtained from naive mice. IgM-/- mice that
received the passive transfer of anti-JHMV Ab had reduced clinical
signs compared with IgM-/- mice that received control Ab
(Table II). Consistent with the reduction
in clinical signs, IgM-/- mice treated with anti-JHMV
Ab also had either undetectable or significantly
(p < 0.01) reduced levels of infectious JHMV
within the CNS (Table II). By contrast, high titers of infectious virus
were detected in IgM-/- mice that received nonimmune
ascites (Table II). Anti-JHMV Ab-protected IgM-/- mice
showed little or no viral Ag in the brain at 21 days p.i. (Fig. 8), while extensive demyelination and
viral Ag in cells morphologically consistent with oligodendrocytes were
found in spinal cords (Fig. 8). By contrast, both the brain and spinal
cord of IgM-/- mice treated with nonimmune ascites had
numerous Ag-positive cells and prominent demyelination (Fig. 8) similar
to untreated JHMV-infected IgM-/- mice (Figs. 3 and 4).
These data support a direct role of the anti-viral Ab response in
controlling the reactivation of JHMV within the CNS and establishing
persistent JHMV infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, most likely secreted by CTL (38, 41, 42), was demonstrated to control JHMV replication in oligodendrocytes
(8), but not neurons (48). Therefore, CTL, either directly via a
perforin-dependent cytolytic mechanism (6) or indirectly via IFN-
release (8), appear to be the predominant effector mechanism for virus
clearance from the majority of infected cells during acute infection.
Similar to infection of IgM-/- mice with CMV (49), LCMV
(9, 50), and influenza B virus (51), the kinetics of viral clearance in
JHMV-infected IgM-/- mice demonstrates that cellular
immunity alone is sufficient to eliminate infectious virus during acute
infection. However, during JHMV infection it is insufficient to control
persistence.
The potential role(s) of humoral immunity has been implicated in
various models of JHMV infection of the CNS. For example, resistant
Brown Norway rats develop a more rapid neutralizing Ab response
compared with susceptible Lewis rats (52). Suckling mice weaned on
immunized dams are protected from acute encephalitis; however,
approximately 40% subsequently develop chronic disease associated with
the evolution of CTL escape variants (24). These data suggest that
partial Ab-mediated protection could, in some instances, predispose the
host to the evolution of viruses able to escape distinct effector
mechanisms. However, during JHMV infection of adult mice, no evidence
for CTL escape mutants was obtained even after reactivation of
infectious virus due to the absence of an Ab response or in the absence
of IFN- (8). The ability of virus-specific mAb to alter JHMV
infection has also been extensively studied. Passive transfer of some,
but not all, neutralizing anti-S protein mAb protect from lethal
challenge and limit immunopathology (25). However, the passive transfer
of nonneutralizing mAb specific for other viral structural proteins is
also protective (26, 27, 28, 29). Limiting infection of neurons and increased
severity of chronic disease were often observed following passive
protection (25, 28). These experiments were generally conducted by mAb
transfer before or concomitant with infection. However, they suggested
that even in those cases in which infectious virus was not reduced, Ab
may play a role in limiting immunopathology. The present study using
IgM-/- mice was unable to confirm a role of humoral
immunity in limiting immunopathology during resolution of acute
infection. Until virus reactivation, there was no difference in the
extent or distribution of mononuclear cell infiltrates or in the number
of apoptotic cells comparing IgM-/- and wt mice. Analysis
of JHMV infection in IgM-/- mice demonstrates an
essential role for humoral immunity in preventing virus reactivation
within the CNS. However, the mechanism(s) via which Ab prevents JHMV
reactivation is not clear. Ab induces a variety of changes within
virus-infected cells that could help establish persistence.
Anti-measles virus and anti-herpes simplex virus Ab not only
reduces surface glycoprotein expression, but also alters viral gene
expression (19, 20). Anti-JHMV Ab prevents both cell death and
cytopathology while selecting for JHMV variants in vitro (53, 54).
Reduced expression of viral glycoproteins has also been observed during
persistent infections with paramyxovirus, retroviruses, rhabdoviruses,
and arenaviruses (55, 56, 57, 58). The immunodominant CTL epitope in
H-2b mice is encoded within the JHMV S glycoprotein (33, 43); therefore, it is possible that interaction between Ab and the S
protein reduces expression of the immunodominant epitope, leading to
viral persistence.
The accumulation of CD8+ T cells within the CNS coincides
with declining infectious virus, and depletion of CD8+ T
cells prevents viral clearance (59, 60). Therefore, CTL appear to be
the major effectors of protective JHMV immunity (6, 7, 38), similar to
many other viral infections (1, 3). There appears to be no relative
reduction of CD8+ T cells or increase in apoptotic cells
within the CNS of the IgM-/- mice during reactivation of
JHMV. Similar to LCMV infection of IgM-/- mice (9, 50),
decreasing peripheral CTL activity was observed in IgM-/-
mice infected with JHMV. Recent data demonstrated a decrease in
Ag-specific IFN- secretion by splenocytes from IgM-/-
mice chronically infected with LCMV (61). Interestingly, the number of
LCMV-specific CD8+ T cells capable of IFN- secretion was
not significantly altered. The defect in IFN- secretion appears more
pronounced in the CD4+ T cell population during LCMV
persistence (61). Increased apoptosis of CD8+ T cells
within the CNS parenchyma is indicative of a defect in CD4+
T cell function during JHMV infection (38). Similar numbers of
apoptotic cells were found within the CNS parenchyma of
IgM-/- and wt mice. In addition, the increased IFN-
mRNA in the CNS during JHMV reactivation suggests that the
CD4+ T cell compartment is not compromised in JHMV-infected
IgM-/- mice. Alternatively, increased IFN- mRNA in the
absence of IFN- secretion could reflect an abortive attempt to
control JHMV reactivation. Although infection with high doses of LCMV
or with a rapidly replicating strain may result in a phenomena termed
CTL exhaustion (9, 50, 62, 63), this mechanism appears unlikely to
account for persistence during an organ-specific infection such as JHMV
infection of the CNS. In contrast to LCMV infection, CD8+
and CD4+ T cells, along with high IFN- mRNA levels, were
detected in the CNS of IgM-/- mice during virus
reactivation. CD8+ T cells exhibiting virus-specific
cytolysis can be activated by in vitro culture of peripheral lymphoid
organs (40, 64). Sequestration, local activation, or expansion during
JHMV infection was previously suggested to account for the absence of
ex vivo CTL activity detected in peripheral lymphoid organs compared
with cells isolated from the CNS (40). Indeed, low level direct
JHMV-specific cytotoxic activity was detected ex vivo using cells
isolated from the CNS of IgM-/- mice at day 35 p.i.
(data not shown). These data suggest that the decrease in splenic CTL
activity may result from sequestration of CTL within the CNS, and argue
against Ag-driven CTL exhaustion as a cause for JHMV reactivation in
the CNS of infected IgM-/- mice.
In the apparent absence of exhaustion, the inability of the CTL to
affect clearance of JHMV in the absence of Ab may reflect an inability
to overcome a normal homeostatic mechanism designed to prevent
excessive immunopathology within the CNS or decreased expression of the
S protein. Indeed, the ability of passively transferred S
protein-specific mAb to protect from JHMV (25) and the ability of
anti-JHMV Ab to facilitate virus persistence in vitro without S
protein-mediated cell-cell fusion (53) support the possibility that
decreased S protein expression facilitates JHMV persistence in the CNS.
However, since CTL effector function requires MHC class I expression,
clearance of virus from neurons and oligodendroglia require additional
effector mechanisms. Indeed, recent data have implicated IFN- as the
major anti-viral effector mechanism controlling JHMV infection of
oligodendroglia (8). The minimal increase in infected neurons in the
IgM-/- mice suggests the possibility that the Ab response
may play an important role in preventing the relatively late spread of
JHMV to neurons, consistent with the analysis of passively transferred
virus-specific mAb before infection (25). Together, these observations
suggest that, in addition to viral cytopathology (3) properties, the
host cells and their phenotypes (15, 16) are also important in
determining the relative importance of the Ab and cell-mediated immune
responses.
In summary, results from the analysis of JHMV infection of the CNS of IgM-/- mice clearly demonstrate that cell-mediated immunity, in the absence of Ab or other related factors, effectively controls virus replication during acute infection. However, in the absence of Ab, infectious virus cannot be effectively cleared from the CNS nor is a persistent CNS infection established. This results in an apparent reactivation of infectious virus and increased mortality despite initial virus clearance from the CNS. These data raise a number of important issues relative to the regulation of immunopathology within the CNS and the mechanism via which Ab suppresses expression of infectious virus within the CNS. These data demonstrate that both cellular and humoral effector mechanisms of the anti-viral immune response play critical, yet apparently distinct roles in the resolution of CNS viral infection.
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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: LCMV, lymphocytic choriomeningitis virus; JHMV, JHM strain of mouse hepatitis virus; N, JHMV nucleocapsid protein; p.i., postinfection; rVV, recombinant vaccinia virus; S, JHMV spike envelope glycoprotein; wt, wild type.
Received for publication October 29, 1998. Accepted for publication March 30, 1999.
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References |
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Interferon is required for viral clearance from central nervous system
oligodendroglia. J. Immunol. In press.
. Lymphokine Cytokine Res. 10:353.[Medline]
in immune cell regulation. J. Leukocyte Biol. 58:373.[Abstract]
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