Chemokine expression in the central nervous system of mice with a viral disease resembling multiple sclerosis: roles of CD4+ and CD8+ T cells and viral persistence.

During the first 45 days after intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV), the levels of mRNAs encoding chemokines MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10 in the central nervous system (CNS) are closely related to the sites of virus gene expression and tissue inflammation. In the present study, these chemokines were monitored during the latter 135 days of a 6-month course of TMEV-induced disease in susceptible (PLJ) or resistant (C57BL/6) mice that possessed or lacked either CD4+ or CD8+ T cells. These data were additionally correlated to mouse genotype, virus persistence in the CNS, antiviral antibody titers, mortality, and the severity of neurological disease. Surprisingly, the major determinant of chemokine expression was virus persistence: the factors of susceptible or resistant genotype, severity of neuropathology, and presence or absence of regulatory T cells exerted minimal effects. Our observations indicated that chemokine expression in the CNS in this chronic viral disorder was intrinsic to the CNS innate immune response to infection and was not governed by elements of the adaptive immune system.

Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler's virus infection.

Central nervous system-infiltrating CD8+ T cells are potential mediators of neuropathology in models of multiple sclerosis induced by Theiler's murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice mount a vigorous cytotoxic T lymphocyte (CTL) response against the immunodominant virus peptide VP2121-130 and clear TMEV infection. Interferon-g (IFN-g)R-/- mice also mount a strong CTL response against the VP2121-130 epitope, but because of genetic deficiencies in critical IFN-g signaling pathways, they do not clear TMEV infection and develop prominent neurological deficits within 6 wk. This pronounced disease process, coupled with a defined CTL response, provides an ideal model for evaluating the importance of antiviral CTL activity in the development of severe demyelination and loss of motor neuron function. By administering the VP2121-130 peptide before and during TMEV infection, 99% of the VP2121-130-specific CD8+ T cell response was inhibited. No decrease in virus infection was observed. Peptide treatment did result in significantly less motor dysfunction, even when no differences in levels of demyelination were observed. Although most investigators focus on the role of CD4+ T cells in demyelinating disease, these studies are the first to demonstrate a clear contribution of antiviral CD8+ T cells in neurological injury in a chronic-progressive model of multiple sclerosis.

The CD4-mediated immune response is critical in determining the outcome of infection using Theiler's viruses with VP1 capsid protein point mutations.

Daniel's strain of Theiler's virus (DA) induces a chronic demyelinating disease in the central nervous system (CNS) of susceptible SJL mice, which serves as an excellent model of multiple sclerosis. We previously demonstrated that point mutations near a putative virus receptor-binding site [VP1 99 (Gly to Ser) or 100 (Gly to Asp)] totally attenuate the ability of DA to persist and induce demyelination in SJL mice. The current studies demonstrate that class II-restricted CD4(+) T cells play a major role in clearing VP1 mutant DA viruses from the CNS to prevent demyelination. Infection of SJL CD4((-/-)) mice with DA-VP1-99(Ser) or DA-VP1-100(Asp) resulted in virus persistence and prominent demyelination in the spinal cord. In contrast, infection of SJL CD8((-/-)) mice with DA-VP1-99(Ser) or DA-VP1-100 did not result in virus persistence or demyelination. In addition, no virus-specific cytotoxicity was observed in CNS-infiltrating lymphocytes following infection of SJL mice with VP1 mutant viruses. The mutant DA-VP1-99(Ser) and DA-VP1(100) viruses were in fact neurovirulent when compared to the wild-type DA virus, as they induced an overwhelming encephalitis and early lethality (2 to 4 days postinfection) in mice deficient in the IFN-alpha/beta receptor. Therefore, the nondemyelinating phenotype observed with DA-VP1-99(Ser) and DA-VP1-100(Asp) viruses is dependent in part on the CD4-mediated host immune response.

HLA-DQ polymorphism influences progression of demyelination and neurologic deficits in a viral model of multiple sclerosis.

The importance of genetic susceptibility in determining the progression of demyelination and neurologic deficits is a major focus in neuroscience. We studied the influence of human leukocyte antigen (HLA)-DQ polymorphisms on disease course and neurologic impairment in virus-induced demyelination. HLA-DQ6 or DQ8 was inserted as a transgene into mice lacking endogenous expression of MHC class I (beta(2)m) and class II (H2-A(beta)) molecules. Following Theiler's murine encephalomyelitis virus (TMEV) infection, we assessed survival, virus persistence, demyelination, and clinical disease. Mice lacking expression of endogenous class I and class II molecules (beta(2)m(o) Abeta(o) mice) died 3 to 4 weeks postinfection (p.i.) due to overwhelming virus replication in neurons. beta(2)m(o) Abeta(o) DQ6 and beta(2)m(o) Abeta(o) DQ8 mice had increased survival and decreased gray matter disease and virus replication compared to nontransgenic littermate controls. Both beta(2)m(o) Abeta(o) DQ6 and beta(2)m(o) Abeta(o) DQ8 mice developed chronic virus persistence in glial cells of the white matter of the spinal cord, with greater numbers of virus antigen-positive cells in beta(2)m(o) Abeta(o) DQ8 than in beta(2)m(o) Abeta(o) DQ6 mice. At day 45 p.i., the demyelinating lesions in the spinal cord of beta(2)m(o) Abeta(o) DQ8 were larger than those in the beta(2)m(o) Abeta(o) DQ6 mice. Earlier and more profound neurologic deficits were observed in beta(2)m(o) Abeta (o) DQ8 mice compared to beta(2)m(o) Abeta(o) DQ6 mice, although by 120 days p.i. both strains of mice showed similar extent of demyelination and neurologic deficits. Delayed-type hypersensitivity and antibody responses to TMEV demonstrated that the mice mounted class II-mediated cellular and humoral immune responses. The results are consistent with the hypothesis that rates of progression of demyelination and neurologic deficits are related to the differential ability of DQ6 and DQ8 transgenes to modulate the immune response and control virus.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

CD4(+) and CD8(+) T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis.

Following intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV), susceptible strains of mice (SJL and PLJ) develop virus persistence and demyelination similar to that found in human multiple sclerosis. Resistant strains of mice (C57BL/6) clear virus and do not develop demyelination. To resolve the controversy about the role of CD4(+) and CD8(+) T cells in the development of demyelination and neurologic deficits in diseases of the central nervous system, we analyzed TMEV infection in CD4- and CD8-deficient B6, PLJ, and SJL mice. Genetic deletion of either CD4 or CD8 from resistant B6 mice resulted in viral persistence and demyelination during the chronic stage of disease. Viral persistence and demyelination were detected in all strains of susceptible background. Although genetic deletion of CD8 had no effect on the extent of demyelination in susceptible strains, deletion of CD4 dramatically increased the degree of demyelination observed. Whereas strains with deletions of CD4 showed severe neurologic deficits, mice with deletions of CD8 showed minimal or no deficits despite demyelination. In all strains, deletion of CD4 but not CD8 resulted in a decreased delayed-type hypersensitivity response to viral antigen. We conclude that each T-cell subset makes a discrete and nonredundant contribution to protection from viral persistence and demyelination in resistant strains. In contrast, in susceptible strains, CD8(+) T cells do not provide protection against chronic demyelinating disease. Furthermore, in persistent TMEV infection of the central nervous system, neurologic deficits appear to result either from the absence of a protective class II-restricted immune response or from the presence of a pathogenic class I-restricted response.

Acceleration in the rate of CNS remyelination in lysolecithin-induced demyelination.

One important therapeutic goal during CNS injury from trauma or demyelinating diseases such as multiple sclerosis is to develop methods to promote remyelination. We tested the hypothesis that spontaneous remyelination in the toxic nonimmune model of lysolecithin-induced demyelination can be enhanced by manipulating the inflammatory response. In PBS-treated SJL/J mice, the number of remyelinating axons per square millimeter of lesion area increased significantly 3 and 5 weeks after lysolecithin injection in the spinal cord. However, methylprednisolone or a monoclonal antibody (mAb), SCH94.03, developed for its ability to promote remyelination in the Theiler's virus murine model of demyelination, further increased the number of remyelinating axons per lesion area at 3 weeks by a factor of 2.6 and 1.9, respectively, but did not increase the ratio of myelin sheath thickness to axon diameter or the number of cells incorporating tritiated thymidine in the lesion. After 3 weeks, the number of remyelinating axons in the methylprednisolone or mAb SCH94.03 treatment groups was similar to the spontaneous remyelination in the 5 week PBS control-treated group, indicating that these treatments promoted remyelination by increasing its rate rather than its extent. To address a mechanism for promoting remyelination, through an effect on scavenger function, we assessed morphometrically the number of macrophages in lesions after methylprednisolone and mAb SCH94.03 treatment. Methylprednisolone reduced the number of macrophages, but SCH94.03 did not, although both enhanced remyelination. This study supports the hypothesis that even in toxic nonprimary immune demyelination, manipulating the inflammatory response is a benefit in myelin repair.

5-Fluorouracil and levamisole exacerbate demyelination in susceptible mice infected with Theiler's virus.

A multifocal inflammatory leukoencephalopathy is associated with the administration of 5-fluorouracil (5-FU), a pyrimidine analogue, and levamisole (LE), an immunomodulator, in patients receiving adjuvant therapy for colon cancer. Cerebral biopsy demonstrated features indistinguishable from multiple sclerosis. We tested whether administration of these agents directly resulted in inflammatory demyelination in mice or whether they exacerbated demyelination in a host predisposed to myelin injury. We used mice intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) which serves as an excellent model for multiple sclerosis. Varying dosages of 5-FU (240 micrograms-2.4 mg) and LE (40 micrograms-1 mg) were administered alone or in combination on a fixed schedule to 52 normal SJL mice and 61 Theiler's virus-infected mice (51 SJL/J mice susceptible to demyelination; 10 C57BL10 mice resistant to demyelination). Controls included 6 noninfected SJL and 26 infected mice (16 susceptible; 10 resistant) treated with phosphate-buffered saline (PBS). Inflammation or demyelination was not detected in brains or spinal cords of noninfected SJL mice treated with 5-FU and/or LE. TMEV-susceptible SJL mice treated with LE alone or in combination with 5-FU demonstrated more extensive inflammation and demyelination at Day 45 than mice treated with PBS. Demyelination was accelerated in infected animals treated with these agents at 45 days but at 70 days a significant difference in extent of demyelination was no longer appreciated between treatment and control groups. Treatment with 5-FU and LE did not convert normally resistant TMEV-infected C57BL/10 mice to demyelination. These experiments support the hypothesis that 5-FU and LE may exacerbate inflammatory demyelination in a susceptible host.

Enhancement of central nervous system remyelination in immune and non-immune experimental models of demyelination.

Studies in both humans and experimental animals indicate that there is potential for full remyelination following CNS demyelination, but the factors that control the degree of myelin repair are unknown. In the Theiler's virus model of demyelination CNS remyelination can be promoted either by global immunosuppression or by selective immunoglobulin therapy. In this paper we discuss whether immunoglobulin-mediated remyelination is a characteristic of immune-mediated demyelination, or whether immunoglobulin-mediated remyelination also occurs in the toxic-traumatic model of lysolecithin-induced demyelination. Our data support the hypothesis that even in a non-primary immune model of demyelination, manipulating the immune system can be beneficial in myelin repair.

The balance between persistent virus infection and immune cells determines demyelination.

We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells.

Theiler's virus persistence and demyelination in major histocompatibility complex class II-deficient mice.

Mice with targeted disruption of the A beta gene of major histocompatibility complex class II molecules (Abo) were used to investigate the role of class II gene products in resistance or susceptibility to virus-induced chronic demyelination in the central nervous system (CNS). Class-II-deficient mice from the resistant H-2b [H-2b(Abo)] and nonmutant H-2b backgrounds were infected with Theiler's murine encephalomyelitis virus intracerebrally and examined for CNS virus persistence, demyelination, and neurologic clinical signs. Virus titers measured by plaque assays showed that 8 of 10 normally resistant nonmutant H-2b mice had cleared the virus within 21 days, whereas the other 2 mice had low titers. In contrast, all class II-deficient Abo mice had high virus titers for up to 90 days after infection (4.30 log10 PFU per g of CNS tissue). Virus antigens and RNA were localized to the brains (cortex, hippocampus, thalamus, and brain stem) and spinal cords of Abo mice. Colocalization identified persistent Theiler's murine encephalomyelitis virus in oligodendrocytes and astrocytes but not in macrophages. There was demyelination in 11 of 23 and 6 of 9 Abo mice 45 and 90 days after virus infection, respectively, whereas no demyelination was observed in infected nonmutant H-2b mice. Demyelinating lesions in Abo mice showed virus-specific CD8+ T cells and macrophages but no CD4+ T cells. Spasticity and paralysis were observed in chronically infected Abo mice but not in the nonmutant H-2b mice. These findings demonstrate that class II gene products are required for virus clearance from the CNS but not for demyelination and neurologic disease.

Gamma interferon is critical for resistance to Theiler's virus-induced demyelination.

Administration of neutralizing monoclonal antibody to gamma interferon increased Theiler's virus-induced demyelination and virus antigen persistence in the spinal cord in susceptible SJL/J mice and completely abrogated resistance such that all C57BL/10SNJ mice developed demyelination. These experiments support the hypothesis that gamma interferon is critically important for resistance to Theiler's virus-induced disease but is not required for myelin destruction.

Promotion of remyelination by polyclonal immunoglobulin in Theiler's virus-induced demyelination and in multiple sclerosis.

Spontaneous remyelination occurs in experimental models of demyelination and in patients with multiple sclerosis, although to a limited extent. This enables the search for factors that promote remyelination. Using the Theiler's virus model of central nervous system demyelination, promotion of remyelination was observed after passive transfer of CNS-specific antiserum and transfer of CNS-specific purified immunoglobulin. Mouse polyclonal immunoglobulin from normal non-syngeneic mice, comparable with the human immunoglobulin preparation, also promotes CNS remyelination in the Theiler's virus model of multiple sclerosis. These experimental findings further bridge the gap with a pilot study that suggests clinical improvement after polyclonal immunoglobulin administration, possibly due to remyelination, in some multiple sclerosis patients with stable, steroid-unresponsive optic neuritis. In view of these experimental and clinical data, the physiological role of myelin in demyelination and remyelination is discussed, and the role of IgG solely as a deleterious molecule in the pathophysiology of multiple sclerosis and experimental demyelination is addressed.

Recombinant human IL-6 suppresses demyelination in a viral model of multiple sclerosis.

We used a murine model of multiple sclerosis (MS) induced by Theiler's murine encephalomyelitis virus (TMEV) to test the effect of IL-6 on central nervous system (CNS) demyelination. Administration of human rIL-6 (2.5 micrograms/dose), beginning one day before infection and then twice daily for 28 days, dramatically reduced demyelination and inflammation in the spinal cord of susceptible SJL/J mice. Benefit also was observed when rIL-6 was used as a therapeutic agent and begun on day 15 after infection, a time in which there is the first evidence of inflammation and demyelination in the spinal cord. Suppression of myelin damage by treatment with rIL-6 was associated with fewer virus Ag-positive cells in the spinal cord. Infectious CNS virus titers, as measured by plaque assay, were reduced in rIL-6-treated animals on day 15 after infection, but not on day 7, 22, or 29 after infection. Total serum Igs and virus-specific Igs, as detected by indirect ELISA, were increased markedly in rIL-6-treated mice, whereas no effect was observed on TMEV-neutralizing Ab titers. In vivo administration of rIL-6 inhibited a murine CNS-demyelinating disease induced by a virus, suggesting that this IL may have application for the treatment of human MS.