key: cord-0005524-vhvnppyb
authors: Kim, Byung Suk; Palna, Joann P
title: Immune mechanisms of Theiler's virus-induced demyelination
date: 1999-09-01
journal: Exp Mol Med
DOI: 10.1038/emm.1999.19
sha: 087c0311aaf0d64d043843372f9a9ccfb853e575
doc_id: 5524
cord_uid: vhvnppyb

nan

Multiple sclerosis (MS) is an immune-mediated neurological disease reflecting demyelination in the white matter of the brain and spinal cord (Adams and Victor, 1977) . The clinical symptoms range from a mild nervous system disability to a severe degenerative paralyzing disorder. Generally, MS is considered to involve autoimmunity to myelin components and is one of the most common neurological disorders of young adults. It is estimated that approximately 300,000 patients are affected in the USA alone. Because of the chronic nature of the disease, social and economic loss by this disease is enormous. Although the cause of MS is unknown, one or multiple infectious agents may be involved in the initial infliction of tissue damage leading to autoimmunity. A possible viral association is suggested by epidemiological studies (Johnson, 1975; McFarlin and McFarland, 1982) as well as the detection of viral antigens and virus-specific antibodies in the majority of MS patients (Soldan et al., 1997) . Several virus-induced and autoimmune models have been used to study the underlying mechanisms of this disease (Daniels et al., 1952; Alford, 1984; Dal Canto et al., 1996; Miller et al., 1997) . These models include demyelinating diseases induced by infection with mouse Hepatitis virus (Lane and Buchmeier, 1997) , Sindbis virus (Griffin et al., 1992) , Semliki Forest virus (Smyth et al., 1990) , Herpes virus (Kastrukoff et al., 1992) , or Theiler's murine encephalomyelitis virus (TMEV) as well as immunization with central nervous system autoantigens. Among these experimental model systems, TMEVinduced demyelination provides an excellent infectious model for the following reasons. The viral structure of TMEV is relatively simple and only few proteins are involved in induction of immune responses. In addition, virus infection has neurotrophism and does not induce any other detec diseases except demyelination. An intracerebral inoculation of TMEV into susceptible strains of mice results in a chronic immune-mediated demyelinating disease that shares many of the features of human MS. For example, chronic pathological involvement is limited to the white matter of the CNS and myelin breakdown is directly related to the clinical symptoms. In addition, demyelination is primarily associated with cell-mediated immune responses and strong autoimmunity to myelin antigens is induced following the initial demyelination by virus-specific T cells .

TMEV is a common enteric pathogen in mice and belongs to the picornaviridae (Theiler and Gard, 1940; Lipton and Friedmann, 1980) . Like other picornaviruses, TMEV has four structural capsid proteins (VP1, VP2, VP3 and VP4) assembled in an icosahedral structure and thus the major immune responses to the virus are against the capsid proteins (Theiler and Gard, 1940; Lipton and Friedmann, 1980) . Two major subgroups of TMEV have been identified: The first subgroup includes GDVII and FA viruses causing rapid and fatal encephalitis and the second subgroup includes the BeAn and DA strains. The second subgroup causes a biphasic neurological disease upon intracerebral inoculation of the virus into susceptible mice . The early, acute phase displays flaccid limb paralysis and degeneration of neurons. The late phase represents chronic, inflammatory demyelination.

Keywords: antigen presentation, multiple sclerosis, infectious immunity-virus, demyelination

Subcutaneous immunization with UV-inactivated TMEV prior to viral infection efficiently protects susceptible mice from demyelinating disease (Yahikozawa et al., 1997) . In contrast, similar immunization of SJL mice after viral infection does not confer protection, but rather accelerate the disease course. These results strongly suggest that the timing of certain immune components is important in the protection and/or induction of demyelinating disease. However, because the virus contains both B cell and T cell epitopes, it is difficult to analyze the individual immune components involved. This diffi-culty is partly overcome by utilizing various fusion proteins containing individual viral capsid proteins or synthetic peptides representing antibody and/or T cell epitope regions. The locations of the major Th epitopes and linear antibody epitopes are diagrammatically shown in Figure 1 .

To dissect the immune components involved in the protection and/or acceleration of demyelination upon immunization with inactivated virus, fusion proteins and synthetic peptides containing the linear antibody epitopes were utilized. Immunization with VP1 and VP2 fusion proteins prior to viral infection, but not VP3, resulted in protection from subsequent development of demyelination ( Figure 2 ). Mice free of clinical symptoms after preimmunization with fusion proteins displayed high levels of antibodies to the capsid proteins. Further immunization with KLH conjugates of synthetic peptides containing individual antibody epitopes indicated that antibodies to only certain linear epitopes are protective, and such antibody-mediated protection appears to be restricted during the early stages of viral infection (Yahikozawa et al., 1997) . This is consistent with previous studies indicating that antibodies to certain viral determinants display strong neutralizing properties in vitro and prevent pathogenesis in vivo following transfusion with antisera (Wada et al., 1994; Kurtz et al., 1995; Sato et al., 1996) .

Treatment with antibodies to either the class II or CD4 molecules suppressed demyelination induced by TMEV (Rodriguez et al., 1986; Welsh et al., 1987) . The majority of MHC class II-restricted helper T cell lines/clones derived from demyelinating lesions of the spinal cords after viral infection reacted with VP1 or VP2 protein, suggesting that these virus-specific Th cells in the CNS are involved in the pathogenicity (Yauch and Kim, 1994) . In addition, the majority (~90%) of T cells recognized one of three predominant epitopes (VP1 233-250 , VP2 74-86 and VP3 24-37 ), one on each major external capsid protein (Yauch et al., 1995) . It is interesting to note that the VP3 Th epitope overlap with the major VP3 linear antibody epitope ( Figure 1 ).

The T cell responses to the three predominant epitopes are similar throughout the disease course without preferential response to any one epitope . However, the level of IFNγ production was highest in response to VP1, intermediate with VP2 and lowest with the VP3 epitope peptide. In contrast, the production of Th2 cytokines is highest in response to VP3 and lowest following stimulation with VP1 epitope peptides. However, it is unclear at this time whether the overlap between Th and antibody epitopes in VP3 influences the type of Th response. Further immunization with peptides demonstrated that VP1 233-250 and VP2 74-86 epitopes are able to exacerbate the disease while VP3 24-37 epitope is not. Levels of various cytokine mRNAs accumulated in the CNS and cytokines produced by T cell clones derived from demyelinating lesions were analyzed (Yauch and Kim, 1994; Palma et al., 1996; Yauch et al., 1998) . Most of the T cell clones produced IL-2 and IFNγ, but not IL-4, suggesting that the majority of Th cells in the CNS is Th1. Interestingly, Th1 cytokine messages preceded Th2 cytokine messages in the CNS of virus-infected SJL mice, suggesting Figure 1. Viral capsid structure and the major epitopes for antibody as well as Th cells are schematically shown. VP1, VP2 and VP3 of Theiler's virus are external capsid proteins. Six major linear antibody epitopes and three Th epitopes have been found. These three Th epitopes, one on each external capsid protein, represent the great majority (~90%) of Th responses to TMEV. Figure 2 . Effect of immune responses to B and Th epitopes on the development of TMEV-induced demyelinating disease. Antibodies to certain epitopes are always protective but some are not. Fusion proteins with both B and T cell epitopes are protective when exposed prior to viral infection. On the other hand, Th responses alone are harmful, regardless of the time of virus exposure. The VP3 region containing both Th and B cell epitopes is an exception to the rule: It does not appear to play an active role in either protection or pathogenesis.

that the initial establishment of Th1 response is critical for the pathogenesis of TMEV-induced demyelination. Taken together, these experiments strongly suggest that the T cell responses to VP1 and VP2 are mainly the Th1 type, and are primarily involved in the pathogenesis of TMEV-induced demyel inating disease (TMEV-IDD).

To understand further the nature of the Th responses, the pathogenic Th1 response to the major VP1 233-250 epitope region has been extensively investigated . The fine epitope regions recognized by individual hybridomas are broad and largely overlapping, but not identical . However, close to 50% of the T cell hybridomas reactive to VP1 233-250 used the T cell receptor Vβ16. In addition, all the T cell hybridomas, recognizing the region with valine at position 245 as the C-terminal residue, expressed Vβ16. Such restriction in the Vα usage among the VP1 233-250specific T cell hybridomas was not found. These results functionally demonstrate for the first time that recognition of the C-terminal residue of a minimal T cell epitope can be associated with a particular Vβ (but not Vα) subfamily-specific sequences, resulting in highly restricted Vβ repertoire of the epitope-specific T cells. Nevertheless, the T cell response to a representative Th1 epitope indicates the broad diversity and complexity of the T cell repertoire towards viral determinants despite the limited number of predominant epitope regions.

The potential role of CD8+ T cells in the pathogenesis of demyelination is controversial. A group of investigators proposed that elimination of myelin producing cells (e.g. oligodendrocytes) are necessary for clinical manifestation and this is mediated by cytotoxic T cells (CTL) which are specific for the virus-infected cells Rivera-Quinones et al., 1998) . However, other groups have demonstrated that treatment with anti-CD8 antibodies does not alter the course of TMEV-IDD (Borrow et al., 1992) . In addition, viral infection can lead to clinical symptoms even in β 2 -microglobulin-deficient mice, lacking functional CD8 + T cells (Pullen et al., 1991; Fiette et al., 1993 ). Moreover, a high level of CTLs is found in resistant mice and a low level in susceptible mice (Dethlefs et al., 1997) . The inconsistent level of virus-specific CTLs in susceptible SJL mice may reflect a poor CD8+ T cell response Lindsley et al., 1991) . Also, adoptive transfer of CD8 + T cells confers resistance to TMEV-IDD in the susceptible recipient mice (Nicholson et al., 1996) , supporting the protective (rather than pathogenic) role of CD8 + CTLs. Furthermore, β 2 -microglobulin-deficient mice with susceptible SJL background are also susceptible to the development of demyelinating disease strongly suggesting that such virus-specific CTLs are not involved in the pathogenesis of demyelination (Yauch and Kim, unpublished observation) . Therefore, the major role of virus-specific CTLs is viral clearance for the protection rather than tissue damage leading to clinical symptoms, in contrast to the published hypothesis (Rivera-Quinones et al., 1998).

The potential involvement of key cytokines for the Th1 response to virus was explored as it is apparently associated with the pathogenesis of demyelination. To examine the potential role of IFNγ, anti-IFNγ antibody was administered to virus-infected mice. These treated mice displayed significantly accelerated disease course and enhanced Th1 responses towards viral antigens (Pullen et al., 1994; Rodriguez et al., 1995) . However, mice that received recombinant IFNγ intracerebrally also demonstrated a similar acceleration of disease. Interestingly, virus-induced demyelination is also accelerated in IFNγreceptor deficient mice, suggesting that IFNγ is not necessary for developing demyelination and perhaps other proinflammatory cytokines such as TNFα may be able to replace the cytokine function (Fiette et al., 1995) . Potential effects of other cytokines on TMEV-induced demyelination have also been investigated. For example, antibodies to TNFα or IL-12 (Inoue et al., 1996; Fushimi et al., 1998; Inoue et al., 1998) , as well as administration of IL-4 (unpublished observation), were able to significantly delay the development and reduce the severity of TMEV-induced demyelination. However, the timing of treatment with respect to viral infection may be critical for the prevention and/or amelioration of disease Bright et al., 1999) . These results strongly suggest that proinflammatory cytokines resulting from the Th1 response to the virus are responsible for the pathogenesis of demyelination, as seen with MS (Benvenuto et al., 1992; Correale et al., 1995; Rieckmann et al., 1995) . Such parallelism between the virus-induc-ed demyelination and human MS makes this system very attractive for studying the underlying mechanisms involved in the maintenance of the Th1/Th2 balance.

Non-pathogenic variant viruses have also been utilized to understand the pathogenic mechanisms instigated by the virus. Several forced mutant viruses resistant to antiviral antibodies or recombinant viruses between viruses resulting in chronic and non-chronic infections have been used to analyze the nature of the virus-induced demyelination (Zurbriggen and Fujinami, 1989; Bureau et al., 1993; Pritchard et al., 1993) . However, these variant viruses are not naturally occurring and thus it is difficult to evaluate the significance of the results. On the other hand, a spontaneous non-pathogenic variant displayed a single substitution of lysine to arginine at position 244 within the VP1 233-250 epitope, which is the only amino acid difference found in the P1 region encoding all of the capsid proteins . In addition, the majority of Th cells specific for VP1 233-250 from mice infected with the pathogenic wild-type virus is Th1 type. Conversely, the major T cell population specific for VP1 K244R from the variant virus-infected mice is Th2 type. Furthermore, the overall T cell response to the variant virus is primarily Th2 type, whereas that induced after infection with the wild type is Th1 type (Figure 3) . Thus, such a spontaneous single amino acid change in a predominant Th epitope of a viral coat protein may exert a profound impact to the type of host immune response and the consequent pathogenesis of virally induced immune-mediated diseases. Moreover, such spontaneous non-pathogenic variants of TMEV may occur more frequently than previously thought.

Using various recombinant viruses, it has previously been established that chronic demyelination is associated with the ability of virus to persist in the CNS (Bureau et al., 1993; Pritchard et al., 1993) . These studies provided valuable information regarding the structural sites involved in viral persistence. However, the location on the viral genome involved in persistence has been controversial. Interestingly, administration of bacterial lipopolysaccharide (LPS), a potent inducer for non-specific inflammatory response, concomitant with viral infection in resistant C57BL/6 mice resulted in enhanced viral persistence, Th responses to viral antigens and subsequent clinical symptoms (Pullen et al., 1995) . Similar enhancement of viral persistence and pathogenicity of a low-pathogenic variant was observed after treatment with LPS (Palma et al., 1996) . These results strongly suggest a possibility that an increase in the inflammatory response in the CNS may prolong viral persistence and further amplify the Th1 response.

Several hypotheses have been proposed to explain virusinduced demyelination. These include: 1) ''bystander'' damage of myelin (Wisniewski and Bloom, 1975; Clatch et al., 1986 ) as a consequence of the inflammatory Th1 response against TMEV antigens, 2) induction of autoimmunity (epitope spreading) to the myelin proteins released by viral damage to the CNS , and/or 3) direct elimination of myelin producing cells (e.g. oligodendrocytes) by cytotoxic T cells (CTL) specific for the virus-infected cells (Rivera-Quinones et al., 1998) . Among these, the ''CTL'' hypothesis is the least consistent with various experimental results as discussed earlier. The ''bystander'' hypothesis favors the involvement of virus-reactive Th1 cells, which activate macrophages resulting in demyelination. The involvement of virus-specific Th1 responses in the pathogenesis of demyelination is supported by strong experimental data. In addition, the fact that autoimmune responses to the major myelin components are followed by the initial inflammatory response to viral antigens supports the potential involvement of ''epitope spreading'' in the pathogenesis of demyelination . However, the degree of contribution of this autoimmunity to the manifestation of overall demyelinating disease is not yet clear, since the clinical signs of demyelination are detected prior to the detection of autoimmunity. In addition, it is difficult to discern at this time that such epitope spreading involves the mechanisms of molecular mimicry with potential cross-reactivity between autoantigens and viral epitopes.

Nevertheless, the local presentation of viral epitopes to infiltrating CD4 + T lymphocytes in the CNS may be a critical step for initiation and propagation of the immunopathologic tissue damage following viral infection (Figure 4) . MHC class II Ags can be expressed on neuroglia cells following exposure to pro-inflammatory cytokines such as IFNγ (Wong et al., 1984) , or virus particles (Massa et al., 1986) . For example, cytokineactivated astrocytes further activate virus-specific, CD4 + T cells and, consequently undergo Fas-mediated apoptosis by the activated T cells in vivo as well as in vitro (Palma et al., 1999) . Since astrocytes play an integral role in maintaining the blood brain barrier (Eddleston and Mucke, 1993; Montgomery, 1994) , such an apoptosis of astrocytes most likely contributes to the pathogenesis of TMEV-induced demyelination. This is consistent with previous studies suggesting the role of Fas- Figure 3 . Potential immunological mechanisms for differential pathogenesis between the pathogenic parental virus and low-pathogenic variant virus. The pathogenic virus contains two Th1-inducing epitopes and one Th2inducing epitope. A mutation in one of theTh1-inducing epitopes converts it to a Th2-inducing epitope tipping the overall balance favoring non-pathogenic Th2 responses.

FasL interaction in multiple sclerosis (Dowling et al., 1996) and similarly in autoimmune mouse models of demyelination (Malipiero et al., 1997; Sabelko et al., 1997; Waldner et al., 1997) . In addition, it is known that activated astrocytes produce a variety of immunoregulatory cytokines (Wesselingh et al., 1990; Lee et al., 1993) , chemokines attracting various inflammatory cells (Tani et al., 1996) , as well as adhesion and co-stimulatory molecules (Shrikant et al., 1995; Nikcevich et al., 1997) . Similarly, virus infection itself may also activate a variety of genes, including chemokines, involved in the immune response. These may promote the influx of inflammatory cells into the CNS further promoting the immunopathological reactions of the CNS leading to immune-mediated demyelination. The initial tissue damage by these virus-specific Th1 cells may facilitate release of abundant CNS autoantigens to the highly inflammatory environment resulting in a vigorous autoimmune response. The ability of direct lysis of the host cells by this virus may also contribute to the release of autoantigens. Therefore, a precise understanding of the pathogenic mechanisms involved in this virus-induced demyelinating disease may provide important general implication regarding the initiation and progression of many different tissue-specific autoimmune diseases such as arthritis and diabetes. . Potential pathogenic mechanisms involved in the immune-mediated demyelination induced following TMEV infection. Resident glial cells may also be activated by inflammatory cytokines and become competent antigen presenting cells that further stimulate inflammatory Th1 cell response in the CNS. Such T cell activation may consequently lead to breaching the blood brain barrier by cytokines and/or apoptosis of CNS cells. The tissue destruction lead to further release of autoantigens facilitating autoimmunity.

Principles of Neurology

Tumor necrosis factoralpha and interferon-gamma synthesis by cerebrospinal fluidderived T cell clones in multiple sclerosis

The role of CD8+T cells in the acute and chronic phases of Theiler's murine encephalomyelitis virus-induced disease in mice

Differential influence of interleukin-12 in the pathogenesis of autoimmune and virus-induced central nervous system demyelination

Mapping loci influencing the persistence of Theiler's virus in the murine central nervous system

Characterization of Theiler's murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEVinduced demyelinating disease: correlation with clinical signs

Patterns of cytokine secretion by autoreactive proteolipid protein-specific T cell clones during the course of multiple sclerosis

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination: a model for human multiple clerosis

Observations on encephalomyelitis of mice

Theiler's virus infection of 129Sv mice that lack the interferon alpha/beta or interferon gamma receptors

The effect of pentoxifylline (PTX) on Theiler's murine encephalomyelitis (TMEV)-induced demyelinating disease

The immune response in viral encephalitis

The level of tumor necrosis factor-alpha producing cells in the spinal cord correlates with the degree of Theiler's murine encephalomyelitis virus-induced demyelinating disease

Suppressive effect on Theiler's murine encephalomyelitis virus-induced demyelinating disease by the administration of anti-IL-12 antibody

The possible viral etiology of multiple sclerosis

Herpes simplex virus type I (HSV I)-induced multifocal central nervous system (CNS) demyelination in mice

Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler's virus: V beta restriction correlates with T cell recognition of the c-terminal residue

A spontaneous low-pathogenic variant of Theiler's virus contains an amino acid substitution within the predominant VP1(233-250) T-cell epitope

B-lymphocyte requirement for vaccine-mediated protection from Theiler's murine encephalomyelitis virus-induced central nervous system disease

Murine coronavirus infection: a paradigm for virus-induced demyelinating disease

Cytokine production by human fetal microglia and astrocytes. Differential induction by lipopolysaccharide and IL-1 beta

Theiler's virus infection of genetically susceptible mice induces central nervous system-infiltrating CTLs with no apparent viral or major myelin antigenic specificity

Cytotoxic T cells isolated from the central nervous systems of mice infected with Theiler's virus

Purification of Theiler's murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence

Myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis is chronic/relapsing in perforin knockout mice, but monophasic in Fas-and Fas ligand-deficient lpr and gld mice

Viral particles induce Ia antigen expression on astrocytes

Multiple sclerosis (first of two parts)

Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading

Astrocytes: form, functions, and roles in disease

Perforindependent neurologic injury in a viral model of multiple sclerosis

Adoptively transferred CD8+ T lymphocytes provide protection against TMEV-induced demyelinating disease in BALB/c mice

IFNgamma-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells

Treatment with lipopolysaccharide enhances the pathogenicity of a low-pathogenic variant of Theiler's murine encephalomyelitis virus

Potential role of CD4+ T cell-mediated apoptosis of activated astrocytes in Theiler's virus-induced demyelination

Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence

Identification of a suitable target cell line for Theiler's murine encephalomyelitis virus specific CTL

Alteration in the level of interferongamma results in acceleration of Theiler's virus-induced demyelinating disease

Treatment with bacterial LPS renders genetically resistant C57BL/6 mice susceptible to Theiler's virusinduced demyelinating disease

Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing-remitting multiple sclerosis is associated with disease activity

Absence of neurological deficits following extensive demyelination in a class Ideficient murine model of multiple sclerosis

Partial suppression of Theiler's virus-induced demyelination in vivo by administration of monoclonal antibodies to immune-response gene products (Ia antigens)

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

Fas and Fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system

A neutralization site of DA strain of Theiler's murine encephalomyelitis virus important for disease phenotype

Intercellular adhesion molecule-1 gene expression by glial cells

Multiplication of virulent and demyelinating Semliki Forest virus in the mouse central nervous system: consequences in BALB/c and SJL mice

Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA

In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis

Encephalomyelitis of mice

Importance of amino acid 101 within capsid protein VP1 for modulation of Theiler's virus-induced disease

Fas-and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis

The effect of L3T4 T cell depletion on the pathogenesis of Theiler's murine encephalomyelitis virus infection in CBA mice

Detection of cytokine mRNA in astrocyte cultures using the polymerase chain reaction. Lymphokine Res

Primary demyelination as a nonspecific consequence of a cell-mediated immune reaction

Inducible expression of H-2 and Ia antigens on brain cells

Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease

Identification of a major T-cell epitope within VP3 amino acid residues 24 to 37 of Theiler's virus in demyelination-susceptible SJL/J mice

A predominant viral epitope recognized by T cells from the periphery and demyelinating lesions of SJL/J mice infected with Theiler's virus is located within VP1(233-244)

Role of individual T cell epitopes of Theiler's virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response

A neutralizationresistant Theiler's virus variant produces an altered disease pattern in the mouse central nervous system

This work was supported by United States Public Health Service Grants, RO1 NS28752, RO1 NS33008, and PO1 NS23349.