Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus

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 x 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.

Fig. 1

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.

Fig. 2

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

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)

Fig. 4

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)

Fig. 5

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.

Fig. 6

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.

Fig. 7

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.