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. Author manuscript; available in PMC: 2017 May 25.
Published in final edited form as: J Neuropathol Exp Neurol. 1999 Jan;58(1):78–91. doi: 10.1097/00005072-199901000-00009

Absence of Spontaneous Central Nervous System Remyelination in Class II-deficient Mice Infected with Theiler’s Virus

M Kariuki Njenga 1,*, Paul D Murray 1,*, Dorian McGavern 1, Xiaoqi Lin 1, Kristen M Drescher 1, Moses Rodriguez 1
PMCID: PMC5444470  NIHMSID: NIHMS859398  PMID: 10068316

Abstract

We previously showed that Theiler’s murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 × 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

Keywords: Axonal damage, Cytotoxic CD8+ T cells, Demyelination, MHC class I, MHC Class II, Remyelination, Theiler’s murine encephalomyelitis virus

INTRODUCTION

Myelin repair in the central nervous system (CNS) is carried out primarily by oligodendrocytes (1–5), but Schwann cells from peripheral nerves can occasionally infiltrate CNS lesions to remyelinate axons (6–8). Theiler’s murine encephalomyelitis virus (TMEV) infection of prototypic susceptible SJL/J mice serves as an excellent model of chronic multiple sclerosis, a disease in which there is progressive damage to myelin lamellae resulting in naked axons with minimal spontaneous repair (9). In certain forms of MS and experimentally induced CNS demyelination, there is extensive repair of demyelinated lesions, indicating that remyelination is a normal physiologic process (10–15). The reasons for the absence of significant repair in some forms of chronic MS, and in SJL/J mice infected with TMEV are not clear. One possibility is that in these diseases, pathogenic components produced within demyelinated lesions damage the axonal surface rendering it incompatible with myelin wrapping (11, 16), or directly inhibit myelin production (5, 17–19). Potential pathogenic factors include the immune inflammatory response (17, 18) and myelin degradation products (5, 19). Alternatively, there may be depletion of endogenous cells or other factors required for myelin synthesis (20–24). For example, in a toxin-induced model of demyelination in rodents, transplantation of glial progenitor cells into demyelinated lesions results in remyelination (22), and treatment with a growth factor promotes synthesis of myelin proteins and proliferation of oligodendrocytes (23, 24), indicating that glial cells and growth factors are required for remyelination. Whatever the factors preventing remyelination in TMEV disease, they are abrogated in TMEV-infected class I-deficient mice, which develop extensive remyelination.

Recent studies have suggested that major histocompatibility complex (MHC) genes play a role in the development of neurologic deficits in chronic central nervous system (CNS) demyelination (25–27). TMEV-infected MHC class II-deficient (Ab°) mice developed both demyelination and neurologic deficits, whereas class I-deficient (β2m [−/−]) mice developed demyelination but no neurologic deficits (25–27). Both mutants were generated from an identical genetic background (C57BL/6 × 129), which is normally resistant to TMEV-induced demyelinating disease. The β2m (−/−) mice had a similar extent and distribution of demyelinated lesions as the Ab° mice, but demonstrated normal spontaneous movement and relatively preserved electrophysiologic activity (27). This absence of neurologic deficits in the TMEV-infected β2m (−/−) mice was associated, in part, with preservation of sodium channel densities and axonal integrity in demyelinated lesions (27), and spontaneous remyelination of 40%, 66%, and 82% of the demyelinated area at 6, 12, and 18 months after TMEV infection, respectively (28). In light of the presence of neurologic deficits in Ab° mice, we investigated the degree of spontaneous remyelination and axonal damage in these mice following TMEV infection.

TMEV-infected class II-deficient mice appear to present a clinical picture that is different from that observed in infected SJL/J mice, the prototypic TMEV-susceptible strain. Infected SJL/J mice, which have extensive axonal disruption (27), develop incontinence, spasticity, and stiffness 4 to 6 months after TMEV infection can survive for up to 15 months with these neurologic deficits, eventually succumbing to severe paralysis and inability to reach food and water (29). In contrast, Ab° mice survive the acute encephalitic phase of TMEV infection (2 weeks), but become spastic and paralyzed as early as 1 month after TMEV infection and die within 2 weeks after onset of these neurologic deficits (30). In addition, not all Ab° mice develop deficits at the same time after infection. Some remain clinically normal for 2 to 3 months before developing neurologic deficits. To understand this high susceptibility of Ab° mice to chronic TMEV infection after surviving the acute encephalitic disease, we examined the temporal clinical phenotype, the integrity of axonal fibers in demyelinated lesions, the capacity for remyelination, and the antiviral humoral and cellular immune responses in these mice.

MATERIALS AND METHODS

Virus

The Daniel’s (DA) strain of TMEV was used for all experiments. The virus was grown in BHK–21 cells and titered by plaque assay in L2 cells as described previously (31). Purified virus for anti-TMEV IgG ELISA and delayed type hypersensitivity (DTH) responses was prepared from infected BHK–21 cells by ultracentrifugation on sucrose and cesium chloride gradient as described previously (31).

Mice

Class II-deficient mice heterozygous for the targeted Ab gene (Ab°) (32) were originally provided by Chris Benoist (Strasbourg, France), whereas class I-deficient mice carrying a deletion in the (β2 microglobulin (β2m) were obtained from R. Jaenisch (Whitehead Institute, Cambridge, Mass.). Both mutants, which are of the same genetic background (C57BL/6 × 129), were bred at the Mayo Immunogenetics Mouse Colony. C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, Me.). Four- to 6-week-old mice were inoculated intracerebrally with 2 × 106 plaque forming units (PFU) of TMEV in a 10 μl volume. Handling of all animals conformed to the National Institutes of Health and Mayo Clinic institutional guidelines.

Methods for Determining Clinical Deficits

For evaluation of clinical neurologic signs, mice were examined 2 times a week and scored using a scale of 0 to 3 in 3 categories: general appearance, activity level, and paralysis. General appearance was scored as follows: 0 = normal appearance, 1 = minimal change in coat or fur, 2 = scruffy appearance, 3 = severely unkempt and incontinent. Activity level scores were assessed as follows: 0 = normal activity, 1 = decreased spontaneous movement, reluctance to move even when tapped, 2 = stiffness, particularly in the hind limbs, 3 = unable to move. Paralysis scores were determined as follows: 0 = no paralysis, 1 = 1 extremity paralyzed, 2 = 2 extremities paralyzed, 3 = 3 or 4 extremities paralyzed. The 3 scores were added to give a composite clinical score (maximum score of 9 for a live mouse). If a mouse died during the experiment, it was given a composite score of 10.

Assessment of Demyelination and Remyelination

Spinal cords were prepared for analysis of demyelination and remyelination as described previously (34). Chronically infected mice were sacrificed by intraperitoneal administration of 10 mg of pentobarbital and perfused by intracardiac puncture with Trumps fixative (100 nM phosphate buffer, pH 7.2, with 4% formaldehyde and 1.5% glutaraldehyde). The entire spinal cord was removed from the spinal canal and sectioned into 1 mm traverse blocks.

To determine the extent of demyelination and remyelination, every third spinal cord block (1-mm-thick) was postfixed in 1% osmium tetraoxide and embedded in araldite (Polysciences, Warrington, Pa.). One micrometer sections were cut and stained with ρ-phenylenediamine. Remyelination was quantitated using Zeiss interactive digital analysis system (ZIDAS) and camera lucida attached to Zeiss photomicroscope (Carl Zeiss Inc, Thornwood, N.Y.). All quantitation was done on coded samples without knowledge of the experimental groups. Ten spinal cord cross sections, spanning the entire spinal cord from cervical to the proximal coccygeal spinal column regions, were examined from each mouse. Total area of the white matter, demyelination, and remyelination were determined for each section using 4×, 10×, and 25× objectives, respectively. For areas of demyelination or remyelination, 3 contiguous axons were required to show the pathologic alteration before the lesion could be outlined for recording. Therefore, individual demyelinated or remyelinated axons, particularly when surrounded nondemyelinated areas, were not included in the analysis. Areas from each of the 10 sections were added to provide total area for each mouse. Areas of demyelination were characterized by cellular infiltration, macrophages engulfing myelin debris, and naked axons. Abnormally thin myelin sheaths relative to axonal diameter, and absence of Schwann cells was used as the criteria for oligodendrocyte remyelination (35). Schwann cell remyelination was identified by thick myelin sheaths, a 1 cell per 1 axon relationship, and the presence of a basement membrane. On average, a typical cross section of 1 mm2 in the lateral column of the spinal cord white matter at the thoracic region contains approximately 300,000 myelinated axons. Selected blocks were trimmed and processed for electron microscopy. Thin sections for electron microscopy were counter-stained with uranyl acetate lead citrate. Statistical comparisons on the extent of demyelination and remyelination were performed using unpaired Student’s t-test.

Assessment of Axonal Integrity

To examine the integrity of axons in the spinal cord white matter of Ab° and β2 m (−/−) mice, we performed immunofluorescence anti-neurofilament and Bielschowski staining independently, as described previously (27). For immunofluorescent staining of neurofilaments, 2 monoclonal antibodies directed against a 68-kDa and a 160-kD peptide were used in concert (Boehringer Mannheim, Indianapolis, Ind.). Paraformaldehyde-fixed frozen sections were incubated for 1 hour with the 2 primary antibodies (2.5 αg/ml of each), followed by incubation with fluorescein isothiocyanate-labeled secondary antibodies. To co-localize white matter areas with inflammatory infiltration, cellular nuclei were visualized using 4,6-diamindino-2-phenylindole (DAPI). The extent of neurofilament disruption in spinal cords was quantitated by using an Olympus Provis AX70 microscope (20× objective) to examine the neurofilament staining patterns in fields of the white matter with inflammatory cellular infiltration from 3 mice of each strain. Photography for neurofilaments and cellular nuclei was performed using a SPOT cooled color digital camera fitted on the Olympus scope. Optics for the appropriate fluorochrome were used to capture registered green (neurofilament) and ultraviolet (cellular nuclear) images.

For Bielschowski staining, adjacent sections were placed in a 20% silver nitrate solution for 15 minutes at 37°C followed by a rinse (27). Sections were then placed in 20% silver nitrate containing ammonium hydroxide for 10 minutes at 37°C and rinsed with distilled water (27). Slides were developed, and washed in ammonium water, 0.2% gold chloride solution for 30 seconds, 5% sodium thiosulphate and tap water before mounting.

Indirect ELISA for Total and TMEV-specific IgG and IgM

Total serum, and TMEV-specific and nonspecific IgG and IgM were determined by ELISA. Mice were bled by cardiac puncture at the time of sacrifice, blood was allowed to clot, and serum was aliquoted and stored at −70°C. For total IgG and IgM, polystyrene microtiter plates (Corning, Corning, N.Y.) were coated overnight with a mixture of goat anti-mouse IgG, IgA, and IgM antibodies (Zymed Laboratories, San Francisco, Calif.) at 4°C. Plates were blocked with 1% bovine serum albumin and sera from individual mice were diluted fourfold (1:500 to 1:128000) in 0.2% bovine serum albumin and incubated on the coated plates for 2 hours at 37°C. To ensure detection of all isotypes, bound IgGs were detected by incubating plates with a mixture of rabbit anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Zymed) at 37°C for 2 hours. IgM was detected using rabbit anti-mouse IgM (Zymed). All rabbit antibodies were of the IgG isotype. Bound IgG or IgM were subsequently detected with alkaline phosphatase conjugated goat anti-rabbit IgG and p-nitrophenylphosphate substrate by reading absorbance at 405 nm. For TMEV-specific IgG and IgM, plates were coated with 0.5 μg of purified TMEV (30). Bound antigens were detected with biotinylated goat anti-mouse IgG and streptavidin conjugated alkaline phosphatase (Jackson Immunoresearch, West Grove, Pa.). p-Nitrophenylphosphate substrate was added and absorbance read at 405 nm. Statistical comparisons of antibody levels between the mutant strains were done at 1:2000 serum dilution using unpaired Student’s t-test.

Virus Plaque and Neutralization Assays

TMEV plaque assays were done on L2 cells as described previously (43). To assess TMEV neutralization by sera from Ab° and β2m (−/−) mice, aliquots of TMEV (200 PFU/ml) were incubated with various dilutions of heat-inactivated sera for 1 hour at room temperature prior to plating onto confluent L2 cells. Sera from noninfected and TMEV-infected C57BL/6 mice were used as negative and positive controls respectively. Data were expressed as serum dilution at which 50% reduction in plaque numbers was observed.

Cytotoxic T-Lymphocyte (CTL) Assay

Mice were sacrificed by ether overdosage and brain and spinal cord aseptically removed without perfusing the mice. Brains and spinal cords from 6 to 10 Ab°, β2m (−/−), or nonmutant C57BL/6 mice infected with TMEV for 7 days were pooled by strain and coded for nonbiased analysis. CNS tissues were homogenized and an aliquot taken for analysis of infectious virus by plaque assay. CNS-infiltrating lymphocytes were isolated by percoll gradient as described previously (36) and resuspended to 2 × 106 lymphocytes per ml in RPMI medium with 5% fetal calf serum. Two-fold serial dilutions were made to provide target-to-effector ratios of between 100:1 and 6.25:1. C57SV cells (H–2Kb and Db) transfected with the LP region of TMEV (leader peptide, VP4, VP3, and VP2) (36), were labeled with sodium [51Cr]chromate (Amersham Life Science Corp, Arlington Heights, Ill.) and served as targets (2 × 104 cells per ml). Equal volumes of target and effector cells were incubated for 5 hours in 96-well round-bottom microtiter plates. The mean radioactivity values were calculated, and standard errors determined from triplicate wells. Untransfected C57SV cells were used as targets to provide background nonspecific lysis. Statistical comparisons for percent virus-specific cytotoxicity were performed by the unpaired Student’s t-test.

RESULTS

Class II-deficient Mice Die Shortly after Onset of Neurologic Deficits

Even though we (26, 27, 30) and others (25) have previously reported the absence of neurologic deficits in β2m (−/−) mice and presence of deficits in Ab° mice, a direct temporal comparison of the 2 mutants, which are from the same genetic background but differ at the MHC loci had not been previously conducted. We therefore detailed the neurologic deficits and mortality and compared these with β2m (−/−) mice. The Ab° mice survived acute TMEV-induced encephalitis (0–21 days postinfection) but developed severe neurologic clinical signs, characterized by decreased spontaneous activity, stiffness of hind limbs, incontinence, and paralysis of 1 or more extremities beginning from day 35 after TMEV infection (Fig. 1A). In a group of 17 Ab° mice monitored twice a week for 130 days, 15 of 17 (88%) developed decreased mobility and scruffy appearance, and 13 of 17 (76%) developed stiffness of posterior limbs or paralysis of at least 1 extremity (Fig. 1A). The Ab° mice presented a heterogeneous clinical picture, with a small number of mice showing clinical disease at any 1 time, and dying 1 to 2 weeks after onset of clinical signs. Ninety-five percent of Ab° mice were dead or had to be sacrificed at a moribund state by 130 days after TMEV infection (Fig. 1B). In contrast, TMEV-infected β2m (−/−) mice and noninfected Ab° mice remained clinically normal with no death for the 4 months of this study (Fig. 1A, B).

Fig. 1.

Fig. 1

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Neurologic disease in class II-deficient (Ab°) and class I-deficient (β2m [−/−]) mice. Mice inoculated intracerebrally with 2 × 106 PFU of DA strain of TMEV were examined twice a week for clinical signs of neurologic disease. Mice were examined for general appearance, spontaneous activity, and stiffness and paralysis of the extremities as described in Materials and Methods. (A) Percent of asymptomatic TMEV-infected Ab° (n = 27), noninfected Ab° (n = 7), and TMEV-infected β2m (−/−) (n = 17) mice. (B) Percent survival of TMEV-infected Ab° (n = 80), noninfected Ab° (n = 7), TMEV-infected β2m (−/−) (n = 12) mice.

Relative Absence of Oligodendrocyte Remyelination in Class II-deficient Mice

Because survival of TMEV-infected Ab° mice was limited to 130 days for 95% of the mice (Fig. 1B), we chose to analyze demyelination and remyelination at day 48 and between day 125 and day 139 after infection by detailed quantitative morphometry. The total area of the spinal cord white matter assessed for demyelination and remyelination was between 7 and 9 mm2 per mouse (Table). The extent of demyelination was similar in both Ab° and β2m (−/−) mice. At 48 days postinfection an average of 0.06 ± 0.04 mm2 of the white matter was demyelinated in Ab° mice as compared with 0.1 ± 0.07 mm2 in β2m (−/−) mice, which was not statistically significant. Between 125 and 139 days after infection, demyelinated lesions had increased seven- to ten-fold in both mutants (Table). The lesions contained abundant macrophages with ingested myelin debris and naked axons.

TABLE.

Absence of Oligodendrocyte Remyelination in Class II-deficient Mice Infected with Theiler’s Virusa

Strain No. of mice Days PIb Total area of white matter (mm2) Total area of demyelination (mm2) Area of demyelination/area of white matter ×100 (%) Total area of remyelination (mm2) Area of remyelination/area of demyelination ×100 (%)
Ab° 5 48 8.0 ± 0.2 0.06 ± 0.04 0.8 ± 0.5 0 0
β2m(−/−) 5 48 6.9 ± 0.6 0.10 ± 0.07 1.3 ± 0.9 0 0
Ab° 8 125–139 8.8 ± 0.4 0.69 ± 0.20 7.4 ± 2.1 0 0
β2m(−/−) 4 125–139 8.7 ± 0.5 0.67 ± 0.20 7.8 ± 2.4 0.1 ± 0.02 14.0 ± 2.0
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a

Data is presented as mean ± standard error.

b

Days PI = days postinfection.

At day 48, there were no measurable areas of oligodendrocyte or Schwann cell remyelination in either mutant. Between 125 and 139 days after infection, 14% of the total demyelinated area was remyelinated by oligodendrocytes in β2m (−/−) mice (Table; Figs. 2B, 3B), whereas there was no oligodendrocyte remyelination observed in Ab° mice (Table; Figs. 2A, 3A). Thin-sheathed myelination characteristic of oligodendrocyte remyelination was extensive in some lesions of (β2m (−/−) mice but was consistently absent in Ab° mice. The absence of oligodendrocyte remyelination in Ab° mice was confirmed by careful electron microscopic study of the lesions (Fig. 3A). Only occasional single axons showed aberrant attempts at CNS remyelination characterized by thin myelin sheaths.

Fig. 2.

Fig. 2

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Light microscopy showing demyelination and remyelination in the spinal cord of class II-deficient (Ab°) and class I-deficient (β2m [−/−]) mice 130 to 140 days after TMEV infection. (A) The Ab° mice developed large demyelinated lesions but minimal if any oligodendrocyte remyelination, whereas (B) demyelinated lesions in β2m (−/−) mice demonstrated oligodendrocyte-medicated remyelination. (Magnification for both pictures ×400)

Fig. 3.

Fig. 3

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Relative absence of remyelination in class II-deficient (Ab°) mice. Electron micrographs showing active demyelinated lesions in Ab° and class I-deficient (β2m [−/−]) mice 130 to 140 days after TMEV infection. (A) Demyelinated axons in the spinal cord of an Ab° mouse. Some oligodendrocytes (o) appear normal morphologically but there is no myelin wrapping of axons. Note multiple macrophages (m) in the lesion with ingested myelin debris. (Magnification ×4.300) (B) Multiple thinly myelinated axons in a lesion from a β2m (−/−) mouse indicative of oligodendrocyte remyelination. An oligodendrocyte (o) remyelinating multiple adjacent axons is shown. Astrocytes (a) which may also be important in remyelination are present in the lesion. (Magnification ×3,500)

In both Ab° (6 of 8) and β2m (−/−) (3 of 4) mice, Schwann cell remyelination characterized by thick-sheathed myelination and 1 cell per internode relationship was scattered and sparse (Fig. 4A, B), especially in large demyelinated lesions. The Schwann cell-type remyelinated lesions occurred at the root entry zones or at the periphery of the cord. Schwann cell remyelination in Ab° and β2m (−/−) mice was not included in the data presented in Table.

Fig. 4.

Fig. 4

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Schwann cell remyelination in both class II-deficient (Ab°) and class I-deficient (β2m [−/−]) mice. Large demyelinated lesions with disruption of the glial limitans appear to attract migration of Schwann cells from the peripheral nervous system to remyelinate CNS naked axons. Schwann cell (s) remyelination characterized by thick myelin sheath and a 1 cell per internode relationship is shown in a demyelinated spinal cord lesions from (A) Ab°, and (B) β2m (−/−) mice. Note presence of macrophages (m) with myelin debris and plasma (p) cells in the lesion. (Magnification for both pictures ×3,900)

Axon Fibers are Disrupted in Class II-deficient Mice

Our previous findings demonstrated that axon fibers are preserved in chronically infected β2m (−/−) mice when compared with SJL/J mice (27). To visualize axon morphology in chronically infected Ab° and β2m (−/−) mice, we used both Bielschowski staining (Fig. 5A, B, C) and immunofluorescent anti-neurofilament staining (Fig. 5D, E, F). Using the DAPI nuclear stain (Fig. 5G, H, I), we examined 6 inflammatory lesions from 3 infected Ab° mice (101 to 135 days postinfection), and 7 similar lesions (based on the level of inflammatory infiltrate) from 3 β2m (−/−) mice (323 days postinfection). This resulted in examination of 2.82 mm2 of white matter in Ab° mice and 6.25 mm2 of white matter in β2 m (−/−) mice. Axonal disruption was identified by the presence of tangled or truncated axons, swelling fibers, and obvious obliteration/degeneration of axonal fibers.

Fig. 5.

Fig. 5

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Bielschowski staining (A, B, C) and anti-neurofilament immunofluorescent staining (D, E, F) of the spinal cord white matter of chronically infected class II-deficient (Ab°) and class I-deficient (β2m [−/−]) mice. In Ab° mice infected with TMEV for 135 days, axons are disrupted (B, E) in regions of inflammatory cell infiltration (H). In contrast, axons are relatively preserved (C, F) in regions of comparable inflammatory cell infiltration (I) in β2m (−/−) mice infected with TMEV for 323 days. The 4,6-diamindino-2-phenylindole nuclear stain was used to select inflammatory lesions in the spinal cord white matter (H, I). Normal axonal distributions (A, D) in the absence of cellular infiltration (G) are shown for uninfected control mouse of identical genotype.

Neurofilament staining revealed disruption and degeneration of axon fibers in white matter inflammatory lesions of Ab° mice (Fig. 5B, E, H). From a total of 28 neurofilament-stained fields examined, 23 (82%) contained axonal disruption, evidenced by tangled filaments, truncated axons, or swollen axon fibers (Fig. 5B, E). In contrast, in lesions with a similar level of infiltration from β2m (−/−) mice, axons were relatively preserved (Fig. 5C, F). Of the 62 fields examined from infected β2m (−/−) mice, 6 fields (10%) had mild axonal swelling and filament tangling, whereas the other 56 fields (90%) had no detectable axonal abnormality. As a control, the axonal fibers and level of cellular distribution in the absence of inflammatory cell infiltration, of age-matched uninfected mice are shown (Fig. 5A, D, G). These results are in agreement with our previous observations in β2m (−/−) mice (27), and suggest that the inflammatory infiltrate in Ab° mice is destructive to axons.

Cytotoxic T Lymphocyte Activity to TMEV Antigens is Present During Acute but not Chronic Infection in Class II-deficient Mice

Neurologic deficits in chronically infected Ab° mice may have been due to depletion or dysfunction of TMEV-specific CTLs, which are present in acute infection (30; Fig. 6). This inability to sustain virus-specific CD8+ T lymphocytes may be the result of selective deletion of cytotoxic T lymphocytes, or lack of helper function by CD4+ T lymphocytes as described previously (37). To investigate this possibility, we analyzed the presence of functional TMEV-specific CTLs in the CNS of chronically infected and reinfected Ab° mice, and compared this with nonmutant C57BL/6. The β2m (−/−) mice could not be used for this comparison because they do not have detectable TMEV-specific CTLs (30). CNS-infiltrating lymphocytes from both Ab° and C57BL/6 mice infected for 7 days showed TMEV-specific cytotoxicity (Fig. 6). At this time (7 days postinfection), the C57BL/6 mice had virus titer of 3.4 × 104 PFU/gm of CNS tissue, whereas the Ab° mice had 2.3 × 106 PFU/gm of CNS tissue. In contrast, both C57BL/6 and Ab° mice had very low levels of functional TMEV-specific CTLs at 50 days postinfection (Fig. 6). The absence of virus-specific CTLs in C57BL/6 mice chronically was expected because they had cleared virus within 21 days postinfection (Fig. 6, data not shown). However, the lack of viral CTL activity in Ab° mice despite 8.6 × 105 PFU of TMEV per gram of CNS tissue indicated inability to sustain the acute CTL response.

Fig. 6.

Fig. 6

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Virus-specific cytotoxic CD8+ T-lymphocytes in the CNS of C57BL/6 and class II-deficient (Ab°) mice. Cytotoxicity against TMEV was assayed against C57SV target cells transfected with TMEV. The results represent activity of pooled CNS-infiltrating lymphocytes from 6 to 10 mice of each strain. The effector to target ratio was 50:1. The level of infectious virus in the CNS at the time of CTL assay (plaque assay) is indicated above each bar. (A) Both strains showed high TMEV-specific cytotoxicity at day 7 postinfection, but not at day 50 postinfection. TMEV titers in C57BL/6 mice were high at 7 days postinfection but undetectable at day 50 postinfection, whereas virus titers in Ab° mice remained high in acute (7 days) and chronic (50 days) disease. Upon reinfection at day 43 postinfection, C57BL/6 mice mounted a strong memory CTL response to clear the virus within 7 days whereas Ab° mice showed no CTL response. (B) Percent lysis of untransfected C57SV cells as controls.

When Ab° mice were infected on day 0, reinfected at day 43, and CTL activity of CNS-infiltrating lymphocytes analyzed on day 50 (7 days after reinfection), no CTL function was detected, and the virus titer was maintained at 2.2 × 105 PFU/gm of CNS tissue (Fig. 6). In contrast, C57BL/6 mice mounted a strong TMEV-specific CTL response (Fig. 6) that cleared the second dose of virus within 7 days. This experiment was performed twice with similar results. The lack of virus-specific CTL response in Ab° mice was not because of lack of CD8+ T lymphocytes in the CNS because CD8+ T lymphocytes but not CD4+ T lymphocytes infiltrated the CNS following TMEV infection (data not shown, 30). We conclude that Ab° mice mount a virus-specific CTL response during acute infection (7 days) but cannot sustain this cytolytic effect in chronic disease.

Class II-deficient Mice have High but Non-virus Neutralizing IgM Antibodies

We have demonstrated previously that antibodies are important in controlling CNS virus replication and spread (38), and may play a role in promoting remyelination (35, 39–41). Therefore, we studied the levels of total nonspecific and TMEV-specific IgG and IgM in chronically infected Ab° mice. The levels of total serum IgG levels in Ab° mice were similar to those observed in nonmutant C57BL/6 mice (p > 0.4) but significantly higher than in β2m (−/−) mice (p < 0.03) (Fig. 7A). This is consistent with other studies that have found decreased levels of circulating IgG in β2m (−/−) mice because of rapid catabolism (42). However, as shown previously, IgG in Ab° mice were not virus-specific (Fig. 7C) (39). In contrast, serum total and TMEV-specific IgM were higher in Ab° mice compared with β2m (−/−) mice (p < 0.014 for total IgM and p < 0.04 for TMEV-specific IgM) (Fig. 7B, D). However, the virus-specific IgM in Ab° mice did not neutralize TMEV in vitro (Fig. 7D insert), but did exhibit wide polyreactivity characteristic of germline natural autoantibodies that promote remyelination (data not shown, 41).

Fig. 7.

Fig. 7

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Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β2m [−/−]) mice. Total and TMEV-specific IgG and IgM in serum of chronically TMEV-infected Ab° (N = 6), β2m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β2m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β2m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β2m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β2m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.

DISCUSSION

In this study, we investigated axonal pathology and spontaneous remyelination in TMEV-infected, class II-deficient mice with an otherwise resistant (H–2b) genotype. Class II-deficient mice demonstrated severe neurologic deficits, axonal disruption and degeneration in 82% of the inflammatory lesions in the spinal cord white matter, and minimal oligodendrocyte remyelination. In contrast, class I-deficient mice of an identical genotype show minimal neurologic deficits and develop CNS remyelination following the demyelinating disease (25, 26, 27).

Based on previous observations that demyelination in chronically infected class I-deficient mice is associated with preservation of axonal integrity and extensive oligodendrocyte-mediated remyelination, we hypothesized that infiltration of CD8+ T cells into demyelinated spinal cord lesions might directly or indirectly damage naked axons, inhibit new myelin formation, and thereby contribute to neurologic deficits (30). In the present study, although we demonstrated CNS infiltration of TMEV-specific CTLs in 7-day infected class II-deficient mice, this response was not detected at 50 days postinfection despite viral persistence. Therefore, if the CNS-infiltrating CD8+ T cells detected in chronically-infected class II-deficient mice are activated and pathogenic to axons or inhibit the remyelination process, they appear to lack specificity for TMEV. Previous experiments in our laboratory have demonstrated the existence of a population of CNS-infiltrating CD8+ T lymphocytes in susceptible strains of mice capable of directly lysing target cells expressing surface antibody to CD3 without further in vitro stimulation (44). Therefore, since CD8+ T cells are one of the primary inflammatory cells infiltrating the CNS of class II-deficient mice (30), these cells may contribute to neurologic injury.

There are several hypotheses explaining the relative absence of remyelination in class II-deficient mice. The first is that a loss of axonal surface integrity renders axons incompatible with the remyelination process. Such changes include aberrant localization of key neurofibrillar proteins (45), alterations in the production of trophic factors or cytokines (46–49), and destabilization of cell membrane by interfering with ion channels (50, 51). Diseases associated with axonal pathologies such as tangling, transection, periaxonal swelling, and myelin rupture, are associated with irreversible neurologic deterioration and the absence of myelin repair (43, 52). A less likely possibility is that a specific component of the class II-restricted immune response is required for remyelination to occur. Although to date, no such cytokine or trophic factor has been implicated, exogenous administration of specific natural autoantibodies directed against unknown antigens on oligodendrocytes promote CNS remyelination in SJL mice chronically infected with TMEV (35, 39–41, 49). In the present study, class II-deficient mice had normal levels of total IgG antibodies, and significantly higher levels of total and virus-specific IgM antibodies, thereby confirming initial findings which demonstrated that class II-deficient mice produced normal levels of all antibody isotypes except IgG1 (32). These data argue against a lack of key natural antibodies as an explanation for the absence of remyelination in class II-deficient mice.

We reported previously that immunosuppression of chronically infected SJL/J (H–2s) mice with antibodies to CD4 increases myelin repair (53). In these earlier experiments, the depletion of CD4+ T cells was conducted late in the disease process (22 to 39 weeks after TMEV infection), and the data were interpreted to indicate that a component of the chronic CD4-mediated immune response to persistent virus inhibits remyelination in SJL mice. However, SJL mice depleted of both CD4 and CD8+ T cells died of overwhelming viral burden (53). Therefore, the pathogenic, demyelination-inducing immune response in SJL mice is balanced by a protective anti-viral immune response. In contrast, in the present study, class II-deficient mice with an otherwise resistant genetic background carry the CD4+ T cell deficiency from birth. Therefore, at every stage of infection, Ab° mice are severely impaired in their ability to generate a protective, class II-mediated antiviral response. Whereas this may prevent the development of some class II-dependent, immune-mediated pathology, the chronic immunodeficiency favors virus replication in the CNS and may contribute to the observed lack of myelin repair.

Alternatively, the relative lack of remyelination may have been influenced by the unique temporal profile for the development of demyelination in Ab° mice. Up to 90 days postinfection, the total areas of white matter demyelination were relatively small, however, the areas increased dramatically by 120 days. Therefore, from the onset of demyelination to the time of sacrifice, there may have been insufficient time for significant remyelination to occur. Unfortunately, the high mortality and severe neurologic deficits observed in Ab° mice made later time points impossible to study.

The mechanism by which class II-deficient mice develop neurologic deficits and rapidly progress to a moribund state is not entirely clear. Although the percent of white matter demyelination in the spinal cord did not correlate with the severity of neurologic deficits in the present study (data not shown), and TMEV-infected SJL/J mice with greater demyelination can survive for up to 15 months (29), it is possible that morbidity and mortality in Ab° mice are the result of discrete lesions developing in vital CNS structures. The inability to control virus via neutralizing antibodies or a sustained CTL response may have resulted in damage to glial cells or neurons. The inability to maintain a CTL response may have resulted from the lack of T cell help from CD4+ T lymphocytes (37), or may have been due to T cell anergy by immune exhaustion (55, 56). Although chronically infected class II-deficient mice had between 105 and 106 PFU of TMEV per gram of CNS tissue throughout the course of disease as compared with less than 103 PFU in class I-deficient and SJL/J mice (53), the reason for the delay in the onset of neurologic deficits and the rapid progression is not clear.

In conclusion, we propose that multiple factors contribute to the lack of remyelination, the onset of neurologic deficits, and rapid progression to death observed in class II-deficient mice. Compared with class I-deficient mice, demyelinating lesions in class II-deficient mice are associated with axonal disruption, lack of remyelination, and the presence of CD8+ T lymphocytes. Class II-deficient mice are unable to generate neutralizing antibodies or maintain detectable virus-specific cytotoxic activity and demonstrate in high viral titers at all stages of the disease. However, unlike T and B cell-deficient mice with the RAG and SCID mutations, which die of neuronal disease within 8–16 days following infection and do not demyelinate (57), class II-deficient mice survive into the chronic stage of disease, suggesting at least a limited ability to control virus replication. Most likely the combination of high virus load and the remaining functioning immune response in class II-deficient mice contributed to the development of neurologic deficits and lack of myelin repair.

Acknowledgments

This work was supported by National Institutes of Health grants NS24180 and NS 32129. M. Kariuki Njenga is supported by National Institutes of Health postdoctoral fellowship 1 F32 NS 10290-01. We thank Mr. and Mrs. Eugene Applebaum and Ms. Kathryn Peterson for financial support for the project.

We thank Mike Coenen, Laurie Zoecklein, Kevin Pavelko, Mabel Pierce, and Sarah Renze for their technical assistance.

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