Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

Abstract

IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4⁺ and CD8⁺ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4⁺ and CD8⁺ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4⁺ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

1 Introduction

IFN-γ is produced by activated NK cells [1], CD4⁺ Th1 cells [2] and CD8⁺ cytotoxic T cells [3] and is a critical component of the cell-mediated immune response to intracellular pathogens and tumors (reviewed in [4]). IFN-γ contributes to activation and differentiation of macrophages [5, 6], T lymphocytes [7, 8] and B lymphocytes [9, 10]. Binding of IFN-γ to its receptor induces transcription of genes for antiviral enzymes [10, 11], contributes to immunity against viruses including HSV-2 [12], MHV [13], and LCMV [14] and plays a role in resistance to a number of intracellular pathogens, including Toxoplasma gondii [15], Leishmania major (reviewed in [16]) and Listeria monocytogenes [17]. Although IFN-γ is not essential for cell survival or tissue homeostasis, the importance of this cytokine is further demonstrated by expression of its receptor in nearly all nucleated cells [18].

Theiler’s murine encephalomyelitis virus (TMEV), a picornavirus, induces a pathological and clinical disease similar to multiple sclerosis [19]. Intracerebral infection with the Daniel strain (DA) of TMEV induces transient, neuronal polioencephalitis followed by chronic white matter demyelination and neurological deficits in susceptible mouse strain such as SJL/J. Resistant mice, such as C57BL/10, recover from the acute disease with no obvious long-term sequelae. Previous studies have demonstrated that both CD4⁺ and CD8⁺ T cells [20–25] are independently required to maintain resistance to TMEV-induced demyelinating disease (TMEV-IDD). IFN-γ has been shown to play a critical role in protection against TMEV-IDD. Monoclonal antibody depletion of IFN-γ abrogates resistance to demyelination in C57BL/10 mice [26] and accelerates and exacerbates demyelinating disease in SJL mice [26, 27]. Similarly, genetically resistant mice with introduced deficiencies in the IFN-γ or receptor genes [28] fail to clear TMEV and develop extensive demyelination and severe neurological deficits following infection with virus.

To investigate which subsets of T lymphocytes must produce IFN-γ to maintain resistance to TMEV-IDD, we used adoptive transfer strategies to create groups of mice in which either CD4⁺ or CD8⁺ T cell populations independently lacked ability to express IFN-γ. To investigate cellular targets of IFN-γ-mediated protection against TMEV-IDD, we used lethal irradiation followed by bone marrow reconstitution to generate mice in which specific populations of cells were deficient in the receptor for this cytokine.

2 Results

2.1 IFN-γ is required for resistance to TMEV-induced demyelinating disease

IFN-γ-deficient mice on an otherwise resistant C57BL/6 background were infected with TMEV and survival was monitored for 6 months (Fig. 1A). IFN-γ^−/− mice experienced 49% mortality (n=37), whereas resistant C57BL/6 mice did not. To measure the development of neurological deficits following infection, rotarod analysis was performed prior to and at multiple time points following infection (Fig. 1B). This assay measures balance and coordination, sensitive and specific indicators of neurological function [29, 30]. During chronic phase of TMEV infection, decline in neurological function in IFN-γ-deficient mice was first detected approximately 45 days post infection and was maximal by approximately 90 days; resistant C57BL/6 mice demonstrated no decline in function. Infectious virus titers of 10³-10⁴ PFU/g of central nervous system (CNS) tissue were detected in IFN-γ^−/− mice throughout course of infection, but not in C57BL/6 mice (Fig. 1C). Viral mRNA was limited to white matter of spinal cord of IFN-γ^−/− mice (Fig. 1D), and was not detectable in resistant C57BL/6 mice (Fig. 1E). Large areas of myelin destruction were found in white matter of IFN-γ^−/− mice (Fig. 1F). Spinal cord lesions contained naked and injured axons, and the normal parenchymal architecture was frequently obliterated by infiltration of foamy macrophages. In contrast, no demyelination was detected in resistant C57BL/6 mice at any time point (Fig. 1G). Measurement of lesion area in IFN-γ^−/− mice (Fig. 1H), demonstrated that at 45 days post-infection, demyelination encompassed 4.4±1.7% of the spinal cord white matter. Demyelination was maximal at 90 days (48.4±4.7%), with no evidence of progression thereafter (34.6±4.1% at 180 days).

Fig. 1.

IFN-γ is required for resistance to TMEV-induced demyelinating disease. (A) Survival in resistant C57BL/6 mice (filled circles) and IFN-γ-deficient mice (open triangles) following infection with TMEV. (B) Decline in rotarod performance in C57BL/6 (black) and IFN-γ-deficient (gray) mice during the chronic demyelinating phase of TMEV infection. Data are presented as ratio of rotarod performance at day 45, 90 and 180 relative to peak performance measured at day 25 post-infection. The 25-day time point was selected because it represents the end of acute encephalitic phase of disease. (C) Titers of infectious virus in CNS were assessed by plaque assay. No infectious virus was detected in C57BL/6 mice (filled circles); however, titers remained high throughout the life of the animal in IFN-γ-deficient mice (open triangles). Data are expressed as log₁₀ PFU/g CNS tissue. (D) In situ hybridization demonstrates location of viral RNA (arrows) in spinal cord white matter of TMEV-infected IFN-γ-deficient mice. (E) Viral RNA was not presented in CNS of resistant C57BL/6 mice. p-Phenylenediamine staining of Araldite-embedded transverse spinal cord sections demonstrates demyelination in (F) IFN-γ-deficient, but not in (G) C57BL/6 mice infected with TMEV for 180 days. (H) Quantitation of demyelination in IFN-γ-deficient mice at 45, 90 and 180 days post infection. Area of demyelination was divided by total area of white matter and expressed as a percent.

2.2 Adoptive transfer model of IFN-γ-dependent resistance to TMEV infection

Although IFN-γ is produced by activated NK cells [1], CD4⁺ T cells [2] and CD8⁺ T cells [3] following virus infections, the relative contributions of lymphocyte subsets to IFN-γ-mediated responses may depend upon specific virus-host interactions. Previous studies have demonstrated that NK cells are not required for protection against TMEV-IDD [31]. To determine the T cell subsets that must produce IFN-γ to maintain resistance to virus persistence and TMEV-induced demyelination, we used adoptive transfer of splenocytes into RAG1-deficient (RAG) mice (Table 1). By using combinations of splenocytes from genetically defined mice, it theoretically would be possible to reconstitute RAG mice with sets of T cells having deficiencies in IFN-γ only in the CD8 or CD4 compartments.

Table 1.

Generation of mice with cell-specific deficiencies in IFN-γ expression^a)

Donor splenocytes	Recipient	New phenotype	Designation
C57BL/6	RAG1−/−	C57BL/6 (IFN-γ+/+)	C57BL/6→RAG
CD4−/− and CD8−/−	RAG1−/−	C57BL/6 (IFN-γ+/+)	CD4+8+→RAG
IFNγ-−/−	RAG1−/−	IFN-γ-deficient CD4+ and CD8+ T cells	IFN-γ−/−→ RAG
CD4−/− and IFN-γ−/−	RAG1−/−	IFN-γ−/− CD4+ T cells	CD4/IFN-γ−/−→ RAG
CD8−/− and IFN-γ−/−	RAG1−/−	IFN-γ−/− CD8+ T cells	CD8/IFN-γ−/−→ RAG

^a)To determine the lymphocyte subsets that must produce IFN- to maintain resistance to virus persistence and TMEV-induced demyelination, we used adoptive transfer of splenocytes into RAG1-deficient (T and B cell-deficient) mice to create mice with cell-specific deficiencies in IFN-γproduction.

To demonstrate the efficacy of the adoptive transfer technique in reconstituting the resistant phenotype, RAG1^−/− mice were reconstituted with splenocytes from resistant C57BL/6 mice (designated C57BL/6→RAG). RAG mice succumb to TMEV infection in 14 days ([32] and data not shown). All seven reconstituted mice survived virus challenge, indicating that adoptively transferred C57BL/6 spleen cells can protect immunodeficient mice from acute viral encephalitis. Similarly, mice reconstituted with spleen cells from C57BL/6 sublines with defective CD4, CD8, IFN-γ, IFN-γ-receptors, or with combinations of these cells, survived the acute encephalitic phase of TMEV infection.

The adoptive transfer model was evaluated to determine whether the chronic phases of disease were faithfully recapitulated. C57BL/6→RAG chimeric mice were compared with C57BL/6 mice for motor function using the rotarod assay. No decline in performance was observed in either group during a 4-month period of observation (Fig. 2A). Histological evaluation of spinal cords from these animals revealed few lesions (Fig. 3A) and no virus was observed in situ within the spinal cord sections (Fig. 4). Together these findings demonstrate that spleen cells from immunocompetent C57BL/6 mice can fully reconstitute the resistant phenotype in otherwise susceptible RAG mice.

Fig. 2.

CD4⁺ T cells make a greater contribution than CD8⁺ T cells to protection against neurological deficits following infection with TMEV. (A) Reconstitution of resistance to TMEV-induced demyelinating disease. We used an accelerated rotarod assay to objectively measure neurological deficits in RAG1^−/− mice which were reconstituted to wild-type phenotypes by adoptive transfer of splenocytes from C57BL/6 mice (C57BL/6→RAG, black bars, n=7), or with a combination of splenocytes from both CD4^−/− (CD8⁺ T cells) and CD8^−/− (CD4⁺ T cells) mice (CD4⁺8⁺→RAG, gray bars, n=6). Each group demonstrated complete preservation of rotarod performance for full 4 months of the study following infection with TMEV. (B) Rotarod analysis in RAG1^−/− mice reconstituted to phenotypes of cell-specific deficiencies in IFN-γ. (IFN-γ^−/−→RAG, black bars, n=5), CD4/IFN-γ^−/−→RAG (gray bars, n=7), and CD8/IFN-γ^−/−→RAG (white bars, n=9) were analyzed for neurological deficits at multiple time points post-infection and results were compared to peak performance. *p<0.05 by ANOVA compared to peak performance.

Fig. 3.

CD4⁺ T cells make a greater contribution than CD8⁺ T cells to IFN-γ-mediated protection against TMEV-induced demyelination. Myelin staining of transverse spinal cord sections from TMEV-infected RAG1^−/− mice reconstituted with spleen cells providing cell-specific deficiencies in ability to produce IFN-γ. No myelin destruction was detected in mice reconstituted to (A) the wild-type phenotype, C57BL/6→RAG (n=7). However, large demyelinating lesions were detected in mice not able to produce IFN-γ in all adoptively transferred spleen cells (B) IFN-γ^−/−→RAG (n=5), (C) transferred CD4 cells, CD4/IFN-γ^−/−→RAG (n=7), and (D) adoptively transferred CD8 cells, CD8/IFN-γ^−/−→RAG (n=9). (E) Areas (mm²) of demyelination in transverse spinal-cord sections were measured and divided by area of spinal cord white matter and expressed as a percentage. * p<0.05 compared with mice reconstituted with C57BL/6 splenocytes by ANOVA on ranks on all groups. ** p<0.05 by ANOVA on mice with demyelination.

Fig. 4.

TMEV persists in mice with cell-specific disruptions in IFN-γ. Immunocytochemistry was used to detect persistent virus-antigen in spinal cord transverse sections of RAG1^−/− mice reconstituted with spleen cells bearing cell-specific deficiencies in ability to produce IFN-γ. Source of reconstituting cells: C57BL/6, no deficiency (n=7); CD4⁺CD8⁺, 1:1 mixture of CD4⁻ and CD8⁻ spleen cells, theoretically should contain complete complement of CD4⁺ and CD8⁺ cells and therefore, no deficiency in their ability to produce IFN-γ (n=6); IFN-γ^−/−, all transferred spleen cells unable to produce IFN-γ (black bar, n=5); CD4/IFN-γ^−/−, a 1:1 mixture of CD4^−/− spleen cells and IFN-γ^−/− cells providing a mixture of cells in which only CD4⁺ cells are not capable of producing IFN-γ (dark gray bar, n=7); and RAGCD8/IFN-γ^−/−-RAG, a 1:1 mixture of CD8^−/− spleen cells and IFN-γ^−/− cells providing a mixture of cells in which only CD8⁺ cells are not capable of producing IFN-γ (light gray bar, n=9). Virus antigen-positive cells were counted and are expressed per mm² of spinal cord white mater. *p<0.05 by ANOVA compared to mice reconstituted with C57BL/6 splenocytes.

The susceptible phenotype was evaluated similarly. Spleen cells from IFN-γ-deficient mice were adoptively transferred into RAG mice yielding IFN-γ^−/−→RAG chimeric animals. Upon challenge with TMEV, these animals developed severe motor function deficits (Fig. 2B) and extensive lesions in their spinal cords (Fig. 3B) during the 4-month observation period. Virus antigen was readily detected in spinal cord sections from these mice (Fig. 4). We conclude from this analysis that both the resistant and susceptible phenotypes are reproduced faithfully using the adoptive transfer model.

Our goal was to analyze animals with cell-specific deficiencies in IFN-γ production to determine whether CD4⁺ or CD8⁺ T cells were primarily sources of protective IFN-γ in this disease model. RAG^−/− mice were reconstituted with splenocytes from CD4-deficient and IFN-γ-deficient mice. The premise behind this protocol is that each type of spleen cell derived from the CD4-deficient mouse would have the capability of producing IFN-γ; however, only CD4⁺ cells present in the mixture would be derived from the IFN-γ-deficient strain and, therefore, would not be capable of producing IFN-γ. This chimera was designated CD4/IFN-γ^−/−→RAG. Similarly, RAG1^−/− mice reconstituted with splenocytes from CD8-deficient and IFN-γ-deficient mice (CD8/IFN-γ^−/−→RAG) would theoretically only contain CD8⁺ T cells incapable of producing IFN-γ. The significant assumption behind this reconstitution protocol is that the immune compartments other than those directly influenced by genetic deletion are intact in each knockout donor cell populations. To evaluate whether this assumption is valid in the context of the TMEV model system, a mixture of spleen cells from CD4^−/−(CD8⁺) and CD8^−/−(CD4⁺) mice was used to reconstitute RAG mice, designated CD4⁺CD8⁺→RAG chimeric mice. According to our hypothesis, chimeric mice containing this mixture of cells should be fully resistant to persistent TMEV infection. As predicted, CD4⁺CD8⁺→RAG chimeric mice developed no motor deficits (Fig. 2A), no demyelinating disease, and virus did not persist in the spinal cord as judged by in situ immunostaining (Fig. 4).

2.3 Both CD4⁺ T cells and CD8⁺ T cells contribute significantly to IFN-γ-mediated protection against chronic demyelination and neurological deficits following infection with TMEV

CD4/IFN-γ^−/−→RAG and CD8/IFN-γ^−/−→RAG chimeric mice display intermediate phenotypes in comparison to resistant C57BL/6→RAG and susceptible IFN^−/−→RAG chimeras. While C57BL/6→RAG chimeras displayed no motor dysfunctions, deficits were progressive beginning at 2 months in the IFN-γ^−/−→RAG and resulted in preservation of only 6.0±4.7% of baseline performance at 4 months post infection. Similarly, impaired function was also detected in CD4/IFN-γ^−/−→RAG mice at 2 months, but decline in function did not progress beyond 50% at 4 months. In CD8/IFN-γ^−/−→RAG chimeric mice, there were no statistically significant differences in rotarod performance from baseline at 2 months, while significant impairment was detected at both 3 and 4 months (65.4+13.2% preserved function at 4 months). Comparison of levels of impairment in CD4/IFN-γ^−/−→RAG and CD8/IFN-γ^−/−→RAG chimeric mice revealed a significantly higher level of impairment in CD4/IFN-γ^−/−→RAG chimeras. These data confirm that IFN-γ is critical in protecting against neurological disease following infection with TMEV and demonstrate that CD4⁺ and CD8⁺ T cell populations each make a discrete contribution to IFN-γ-mediated protection against TMEV-induced neurological deficits.

Differences in motor function seen in the CD4/IFN-γ^−/−→RAG and CD8/IFN-γ^−/−→RAG chimeric mice were associated with differences in extent of demyelination. CD4/IFN-γ^−/−→RAG chimeric mice were significantly more demyelinated than were CD8/IFN-γ^−/−→RAG chimeric mice (Fig. 3E). As expected, TMEV antigen was readily detectable in spinal cords from chronically infected CD4/IFN-γ^−/−→RAG and from CD8/IFN-γ^−/−→RAG chimeric mice (Fig. 4). Virus antigen was localized entirely to the white matter of spinal cord. There were no statistically significant differences in virus antigen-positive cells/mm² between the groups of mice with IFN-γ deficiencies.

2.4 IFN-γ mediates resistance to TMEV by acting on irradiation resistant cells from the host and does not need to act on bone marrow-derived cells in bone marrow chimeras

To determine which sets of cells must express IFN-γ receptor for IFN-γ to mediate resistance to virus persistence and TMEV-induced demyelination, we generated mice with cell-specific deficiencies in expression of the receptor for IFN-γ (IFN-γR) (Table 2). Previous in vivo and in vitro studies have demonstrated that IFN-γR^−/− mice develop normal type 1 T cell responses, as well as normal IL-12 and IFN-γ production in response to antigen [33, 34]. However, IFN-γ influences the differentiation of T and B lymphocytes by promoting the Th1-inducing effects of IL-12 [7, 8] and enhancing isotype switch to IgG2a [9, 10]. Therefore, it was of interest to determine whether IFN-γ was acting on lymphocytes or on other cell types (presumably virus-infected cells in the CNS) in this model of anti-viral immunity. When splenocytes from mice deficient in IFN-γR were adoptively transferred into RAG1-deficient mice, only T cells and B cells in these IFN-γR^−/−→RAG chimeric mice lacked the ability to express the IFN-γR. Alternatively, lethal irradiation followed by bone marrow reconstitution was used to generate mice with cell-specific deficiencies in the expression of the IFN-γR. In lethally irradiated RAG1^−/− mice reconstituted with bone marrow from mice lacking the receptor for IFN-γ, IFN-γR was not expressed on bone marrow-derived cells (T cell, B cells, NK cells, and macrophages), but expression of IFN-γR on irradiation-resistant cells in the host animal was unimpaired. This group of animals was designated IFN-γR^−/−→RAG. Similarly, lethally irradiated IFN-γR^−/− mice were reconstituted with bone marrow from wild-type, resistant B6×129 mice. These chimeric mice, designated B6×129→IFN-γR^−/−, lacked IFN-γR expression on host cells, including key cell types in CNS at site of infection, such as oligodendrocytes, neurons and microglia. However, expression by bone marrow-derived lymphocytes in these mice was normal.

Table 2.

Generation of mice with cell-specific deficiencies in IFN-γR expression^a)

Donor	Recipient	New phenotype	Designation
IFN-γR-deficient splenocytes	RAG1−/−	IFN-γR-deficient splenocytes	IFN-γR−/−→ RAG
IFN-γR-deficient Bone marrow	RAG1−/− (lethally irradiated)	IFN-γR-deficient CD4+ and CD8+ T cells, B cells, NK cells, macrophages, etc.	IFNγR−/−→ RAG bone marrow chimera
129×C57BL/6 F1 Bone marrow	IFNγR−/− (lethally irradiated)	IFN-γR-deficient host cells, including neurons, oligodendrocytes, and parenchymal microglia	129×C57BL/6 F1 IFN-γR−/− bone marrow chimera

^a)We sought to determine the cellular subsets that must express the receptor for IFN-γ to maintain resistance to virus persistence and TMEV-induced demyelination. We used adoptive transfer of splenocytes into RAG1-deficient mice as well as bone marrow reconstitution of lethally irradiated mice to generate groups with cell-specific deficiencies in the receptor for IFN-γ.

There were no statistically significant declines in rotarod performance at 2 or 3 months post infection (p>0.05 by repeated measures ANOVA) in IFN-γR^−/−→RAG chimeric mice (Fig. 5A). Pathological analysis demonstrated that these mice maintained complete resistance to TMEV-induced demyelination in spinal cord white matter (n=7) (Fig. 5B). As these animals lack IFN-γR expression only in bone marrow-derived cells, the mechanism of IFN-γ-mediated protection against TMEV-induced demyelination and neurological deficits does not require direct binding of IFN-γ to its receptor on these cells. IFN-γR^−/−→RAG bone marrow chimeras were also resistant to TMEV-induced demyelinating disease (Fig. 6A). These mice displayed the same phenotype as (129×C57BL/6) F1 hybrid mice (Fig. 6B). In contrast, demyelinating lesions were detected in all 129×C57BL/6→IFN-γR^−/−mice (Fig. 6C), with an average demyelination of 7.6±3.4% of the spinal cord. We conclude that the receptor for IFN-γ must be present on irradiation-resistant host cells for IFN-γ to mediate protection against TMEV virus and associated demyelinating disease.

Fig. 5.

Mice lacking the IFN-γ receptor on T and B lymphocytes are resistant to TMEV-induced neurological deficits and demyelination. T and B cell-deficient RAG1^−/− mice were reconstituted with splenocytes from mice genetically deficient in receptor for IFN-γ. (A) IFN-γR^−/−→RAG mice were assessed monthly for development of neurological disease using an accelerated rotarod assay. There were no statistically significant decline in rotarod performance at 2 or 3 months post-infection (p>0.05 by repeated measures ANOVA). (B) Araldite-embedded transverse spinal-cord sections demonstrated normal myelin with no demyelination (n=7).

Fig. 6.

The receptor for IFN-γ must be present on irradiation resistant cells from the host, but not bone marrow-derived lymphocytes, to maintain resistance to TMEV-induced demyelination. Myelin staining of transverse spinal cord sections from chimeric mice infected with TMEV for 4 months. No demyelination was detected in (A) wild-type B6×129 mice (n=8) or (B) from IFN-γR^−/−→RAG bone marrow-chimeric mice (n=11) that lack the receptor for IFN-γ on bone marrow-derived cells. (C) Demyelinating lesions were detected in RAG→IFN-γ^−/− bone marrow-chimeric mice that lack receptor for IFN-γ on all host cells, including CNS glia (n=5). (D) Areas (mm²) of demyelination in transverse spinal cord sections from RAG→IFN-γ^−/− bone marrow-chimeric mice were measured and divided by area of spinal cord white matter and expressed as a percentage (n=5).

3 Discussion

We and others have demonstrated that IFN-γ is critical for maintaining resistance in vivo to chronic demyelination following infection with TMEV [26–28]. Here we show that IFN-γ production by both CD4⁺ and CD8⁺ T cell subsets is required for resistance to TMEV-induced demyelinating disease. Using adoptive transfer techniques, we generated mice with an otherwise resistant genetic background in which either CD4⁺ or CD8⁺ T cell population lacked the ability to secrete IFN-γ. In either group, infection with TMEV resulted in viral persistence, demyelination and development of neurological deficits. Disease was less severe than in mice in which both populations of cells lacked the ability to express IFN-γ and CD4⁺ T cells appear more important determinants of resistance than CD8⁺ T cells in our experiments.

The significance of IFN-γ production by CD8⁺ and CD4⁺ T cells may vary during infection with different viruses and pathogens. For example, in a transgenic model of hepatitis B infection, adoptively transferred virus-specific CTL completely abolished HBV gene expression and replication in and IFN-γ-dependent manner [35]. Similarly, in LCMV infection, CD8⁺ T cells are preferentially expanded and account for 60-90% of the IFN-γ-producing lymphocytes [36]. In contrast, IFN-γ derived from CD4⁺ T cells, but not NK or CD8⁺ T cells is critical for activation of infected macrophages and resistance in experimental leishmaniasis in mice (reviewed in [16]). Similarly, in experimental leishmaniasis, although IFN-γ-production by NK cells can influence early parasite burden, and CD8⁺ T cells contribute to IFN-γ production during re-infection of mice previously cured of the disease, only CD4⁺ T cell-derived IFN-γ is critical for resistance (reviewed in [16]). Therefore, variation in pathogen tropism may influence IFN-γ-mediated responses of the host.

Protection against demyelinating phase of disease does not appear to require direct IFN-γ-mediated modulation of T cell or B cell effector function. IFN-γ contributes to the recruitment and activation of lymphocytes, enhances Th1 differentiation [7, 8], and influences the B cell isotype switching [9, 10]. In the current studies, mice in which receptor for IFN-γ was absent from T cells and B cells or from all hematopoietic cells maintained resistance to TMEV-induced demyelinating disease. We therefore concluded that IFN-γ-mediated protection is not dependent on its interaction with any of these cell populations. In contrast, mice that lacked IFN-γR on non-hematopoietic host somatic cells developed demyelination. This finding demonstrates that the receptor for IFN-γ must be present on non-lymphoid cells in order to maintain resistance to TMEV-induced demyelination. These data support the hypothesis that IFN-γ-mediated protection against viral persistence and demyelination is due to a direct effect on infected cells in the CNS.

Yap and Sher [34] used bone marrow chimeric mice to evaluate the requirement for IFN-γR on hematopoietic versus nonhematopoietic cells in resistance to Toxoplasma gondii and Listeria monocytogenes infection in mice. T. gondii infects hematopoietic, neuronal, epithelial and mesodermal cells, and IFN-γR expression was required on both hematopoietic and somatic cells for acute resistance. Another example of direct IFN-γ-mediated protection from a pathogen involves resistance to macrophage-trophic intracellular bacterium Listeria monocytogenes. Because IFN-γ-mediated protection requires activation of infected macrophages [16], it is not surprising that in this model resistance requires IFN-γR expression only on cells derived from the bone marrow [34].

The experiments reported here support a model in which T lymphocyte subsets play distinct roles in IFN-γ-mediated protection from TMEV-induced demyelination disease. T cell secretion of IFN-γ may result in direct neuroprotection of CNS glia by either up-regulating anti-viral enzymes [37] or by augmenting antigen presentation through the up-regulation of MHC in the CNS. In acute TMEV infection, MHC class I-restricted cytotoxic T cells are important in protecting against subsequent development of chronic demyelination [20]. Concomitant with lysis of virally infected cells, paracrine secretion of IFN-γ would increase MHC class I expression and up-regulation of antiviral enzymes on neighboring cells within the CNS [4, 37]. This would enhance immune recognition of virally infected cells, while simultaneously protecting cells not yet infected. In addition, interaction between MHC class II-restricted CD4⁺ T cells and perivascular microglia, the primary antigen-presenting cells of the CNS, may be critical to the amplification of immune responses within the CNS. IFN-γ from CD4⁺ T cells activates monocytes and microglia in the CNS. Increased expression of MHC class II and costimulatory molecules may be needed for continued activation of CD4⁺ T cells. In addition, a robust T helper response is required for maintaining cytotoxic responses of CD8⁺ T cells. This model provides an explanation as to how cytokine production by discrete lymphocyte populations can have independent effects on an acute antiviral responses, and is consistent with previous experimental data relating to antigen presentation, lymphocyte function and cytokine expression within the central nervous system.

4 Materials and methods

4.1 Virus

The Daniel strain of TMEV was used for all experiments. The passage history has been described previously [38, 39].

4.2 Mice

C57BL/6 (the prototypic resistant strain), C57BL/6-IFN-γ^tm1Ts, C57BL/6-Rag1^tm1Mom, C57BL/6-CD4^tm1Mak, C57BL/6-CD8^tm1Mak, B6, 129-Rag1^tm1Mom, 129-IFN-γR^tm1, and C57BL/6-IFN-γ^tm1Ts mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice from 4 to 8 weeks of age were injected intracerebrally with 2×10⁶ PFU of virus in a 10 μl volume. Handling of all animals conformed to the National Institutes of Health and Mayo Institutional guidelines.

4.3 Reconstitution of RAG1-deficient mice by adoptive transfer

Donor spleen cells (3×10⁷ in 0.3 ml) were injected i.v. into the tail vein of recipient mice. This number of cells protects SCID mice from fatal neuronal encephalitis or chronic white matter demyelination following TMEV infection [40].

4.4 Bone marrow transplantation

Recipient mice were irradiated with 950 rad and reconstituted intravenously with 5×10⁶ donor bone marrow cells. Mice were treated with tetracycline (0.12 g/l) in the drinking water for 1 week prior to irradiation, and for 1 week following irradiation. Mice that were not reconstituted with bone marrow died in 12 or 13 days. Three months following bone marrow transplant, mice were infected with TMEV.

4.5 Clinical assessment of disease using an accelerated rotarod assay

The Rotamex rotarod (Columbus Instruments, Columbus, OH) measures balance, coordination, and motor control and was used to assess neurological function in this study. The rotarod consists of a suspended rod powered by a variable speed motor capable of running at a fixed speed or accelerating a constant rate. Mice were trained and tested according to the protocol established by McGavern et al. [29]. Mice were evaluated using an accelerating assay (initial speed of 10 rpm accelerating at 10 rpm/min until the mouse fell off). Rotarod performance was measured each month following virus infection. The change in rotarod velocity that occurred prior to the mouse falling was recorded for each mouse, and data were expressed as percent decrease from baseline.

4.6 Viral plaque assay

Viral titers in CNS homogenates were determined as previously described. Briefly, a 10% (w/v) CNS homogenate was prepared in DMEM and clarified by centrifugation. All plaque assays were performed in duplicate and without knowledge of mouse identity.

4.7 In situ hybridization for viral RNA

In situ hybridization for TMEV RNA was conducted as described previously [24]. Slides were hybridized with ³⁵S-labeled 363-bp (nucleotides 3306–3668) cDNA probes corresponding to the VP1-coding sequence of TMEV (DA strain).

4.8 Immunohistochemistry for viral antigen

Saggital spinal cord blocks (12–15 blocks per mouse) were embedded in paraffin for immunohistochemistry. Slides were deparaffinized in xylene, the rehydrated though a series of ethanol rinses (absolute, 95%, 70% and 50%) prior to the addition of the primary antibody. Spinal cord sections were incubated with a polyclonal rabbit-antiserum to purified DA TMEV that specifically reacts to all structural proteins of TMEV [38]. Slides were incubated with a biotinylated secondary antibody and detection was performed using the avidin-biotin immunoperoxidase system (Vector Laboratories, Burlingame, CA). Slides were developed using Hanker-Yates reagent (Polysciences, Warrington, PA). Quantitative analysis was performed using a Zeiss microscope attached to a camera lucida on a ZIDAS (Carl Zeiss Inc., Oberkochen, Germany) digitizing tablet. Spinal cord areas were traced to determine total area (mm²). The number of virus antigen-positive cells for each mouse was counted and expressed per mm² of the spinal cord white matter.

4.9 Preparation of spinal cord tissue and analysis of neuropathology

The preparation of spinal cords for histological analysis has been described previously [26, 31, 33]. Every third block (12–15 blocks/mouse) was osmicated, embedded in Araldite (Polysciences) and stained for myelin using p-phenylendiamine (Sigma, St. Louis, MO). A Zeiss microscope attached to a camera lucida was used to project the spinal cord images onto a ZIDAS (Carl Zeiss) digitizing tablet. Spinal cord areas were traced to determine area (mm²) of white matter, demyelination, and remyelination. Demyelinating lesions were characterized by cellular infiltration, macrophages engulfing myelin debris, and presence of naked axons. Area of demyelination was divided by area of white matter and expressed as a percent. Area of remyelination was divided by area of demyelination and expressed as a percent.

Abbreviations

TMEV

Theiler’s murine encephalomyelitis virus

DA

Daniel strain

TMEV-IDD

TMEV-induced demyelinating disease

RAG mice

RAG1-deficient mice

CNS

Central nervous system