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The Journal of Immunology, 2002, 168: 4221-4230.
Copyright © 2002 by The American Association of Immunologists

Preferential Induction of IL-10 in APC Correlates with a Switch from Th1 to Th2 Response Following Infection with a Low Pathogenic Variant of Theiler’s Virus1

JoAnn P. Palma2, Robert L. Yauch2,3, Hee-Kap Kang, Hee-Gu Lee4 and Byung S. Kim5

Departments of Microbiology-Immunology and Pathology, Northwestern University Medical School, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Theiler’s murine encephalomyelitis virus induces immune-mediated demyelination in susceptible mice after intracerebral inoculation. A naturally occurring, low pathogenic Theiler’s murine encephalomyelitis virus variant showed a single amino acid change within a predominant Th epitope from lysine to arginine at position 244 of VP1. This substitution is the only one present in the entire viral capsid proteins. In this paper, we demonstrate that the majority of T cells specific for VP1233–250 and VP274–86 from wild-type virus-infected mice are Th1 type and these VP1-specific cells poorly recognize the variant VP1 epitope (VP1K244R) containing the substituted arginine. In contrast, the Th2-type T cell population specific for these epitopes predominates in variant virus-infected mice. Immunization with UV-inactivated virus or VP1 epitope peptides could not duplicate the preferential Th1/Th2 responses following viral infection. Interestingly, the major APC populations, such as dendritic cells and macrophages, produce IL-12 on exposure to the pathogenic wild-type virus, whereas they preferentially produce IL-10 in response to the low pathogenic variant virus. Thus, such a spontaneous mutant virus may have a profoundly different capability to induce Th-type responses via selective production of cytokines involved in T cell differentiation and the consequent pathogenicity of virally induced immune-mediated inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Theiler’s original viruses, including the BeAn 8386 and DA strains (1, 2, 3), can cause a chronic biphasic neurological disease on intracerebral inoculation into susceptible mice (2, 4, 5). In particular, the BeAn strain is known to induce a very mild early phase disease, although it manifests a clinically severe late phase white matter disease resulting in spastic waddling gait, extensor spasms, and incontinence (2). Persistent Theiler’s murine encephalomyelitis virus (TMEV)6 infection results in the development of a chronic, immune-mediated inflammation in the CNS (6). Various parameters associated with this disease parallel those of human multiple sclerosis; thus, this system is considered to be an important infectious model for multiple sclerosis (6, 7).

It has been well established that cytokines produced by Th2-type cells inhibit the phagocytic ability of activated macrophages as well as the production of inflammatory cytokines by Th1-type cells involved in a variety of immune-mediated diseases (reviewed in Ref. 8). Certain Th2 cytokines (e.g., IL-4 and IL-10) are also known to inhibit inflammatory Th1 responses in vivo (9, 10, 11, 12). The reciprocal regulation of Th1 and Th2 responses is critical for the resolution or progression of many infectious diseases (8, 13). In addition, Th2 responses involving the production of IL-4 and IL-10 can suppress the development of CNS inflammation associated with autoimmune demyelination, experimental autoimmune encephalomyelitis (EAE) (14, 15, 16), although a single cytokine effect may not be sufficient. Moreover, recovery from EAE is associated with the presence of Th2-type cytokines (17, 18). Aside from the direct down-regulation of pathogenic Th1 responses, Th2 cytokines can also exhibit protective effects against neuronal cell injury caused by activated microglia (19, 20). Thus, the Th1/Th2 balance appears to be very important for the induction/maintenance of inflammatory demyelinating disease in the CNS.

In general, the Th1-type response is efficient in the protection from various viral infections. In contrast, several lines of observation strongly suggest that Th1 response to viral Ag is critically important for the development of TMEV-induced demyelinating disease (21, 22, 23). In addition, treatments with Abs to various cytokines involved in inflammatory Th1 responses effectively reduced the demyelinating disease (7, 24, 25), strongly supporting the importance of Th1 response for pathogenesis. The major T cell populations specific for TMEV during the course of disease recognize three predominant viral epitopes (VP1233–250, VP274–86, and VP324–37), one each on the external capsid proteins (21, 22, 26). The T cell populations specific for VP1 and VP2 epitopes in the demyelinating lesions of the CNS are primarily the Th1 type. These T cells appear to be responsible for the development of immune-mediated demyelination because immunization with these epitope peptides resulted in acceleration of the disease (23). However, the mechanisms involved in the initiation of such Th1 responses on viral infection are not yet known.

We have recently isolated a spontaneously occurring TMEV variant (M2) that exhibits low pathogenicity in susceptible SJL/J mice (27). Interestingly, T cells specific for the predominant T cell epitope (VP1233–250) of VP1 capsid protein of the parental virus react poorly with the variant virus containing a single amino acid substitution within this epitope. However, pre-exposure of susceptible SJL/J mice to this variant virus results in a strong protective immunity against subsequent infection with the pathogenic virus. The levels of Abs to the virus as well as to the major linear epitopes were similar in mice infected with either the variant or wild-type virus. Only a single substitution of lysine to arginine at position 244 of the predominant VP1 Th epitope was found within the entire P1 region encoding all of the viral capsid proteins (27). Such an alteration at the Th epitope in the spontaneous variant is in sharp contrast to the nonpathogenic variants selected for resistance to antiviral Abs, exhibiting amino acid substitutions at non-Th epitope regions, probably the Ab sites (28, 29). Thus, analysis of the immune response to naturally occurring, low pathogenic variants may provide important insights into the viral pathogenesis of immune-mediated demyelination.

To understand the pathogenic mechanisms involved in the immune-mediated inflammatory demyelination, the nature of T cell responses to this low pathogenic variant virus was compared with that of pathogenic virus. We report here that this single amino acid substitution in a capsid protein of the low pathogenic variant leads to the development of noninflammatory Th2 response to the VP1 epitope and other predominant Th epitopes as well as whole variant virus, in contrast to Th1 responses by the pathogenic parental virus. Further immunization experiments using predominant epitope peptides indicated that skewed Th2 response induced by the variant is not due to the property of the variant epitope. However, the pathogenic wild-type and nonpathogenic mutant viruses differentially induce IL-12 and IL10 in isolated macrophages and dendritic cells (DC) after viral infection, respectively. These results strongly suggest that the underlying differences in pathogenicity between the wild-type and variant virus perhaps lie in the differential induction of Th1/Th2 response influenced by the cytokines produced in APC during the initial viral infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female SJL/J mice, 4–6 wk old, were purchased from either The Jackson Laboratory (Bar Harbor, ME) or the Charles River Laboratories (Boston, MA) through the National Cancer Institute (Bethesda, MD).

Viruses

The parent BeAn 8386 (173R) stock and nonpathogenic (M2) viruses derived from the parent stock were propagated in BHK-21 cells in DMEM supplemented with 7.5% donor calf serum. Virus was purified by isopyknic centrifugation on Cs2SO4 gradients as previously described (30). TMEV was inactivated by UV irradiation, and inactivation was verified by the inability of the virus to produce plaques on BHK-21 cells.

Synthetic peptides

The synthetic peptides representing the amino acid residues of TMEV were prepared using the RaMPS system (DuPont, Wilmington, DE) with 9-fluorenylmethyloxycarbonyl reagents. A major single peptide (>95%) was present in each of the peptide preparations based on the reverse phase HPLC analyses.

Infection and immunization of mice with TMEV

Varying concentrations of virus in 30 µl DMEM were administered intracerebrally in the right cerebral hemisphere of mice anesthetized with methoxyflurane. This inoculum of pathogenic parental virus stock consistently induced chronic gait abnormality and neurological signs (>90% of mice) in SJL/J mice (31). For immunization, mice were injected s.c. in the base of the tail with 100 µl of a 1:1 emulsion of UV-inactivated wild-type or M2 virus (50 µg) in CFA. Nine days later, lymph node (LN) cells were pooled from two to three mice, and the level of T cell proliferation was subsequently assessed in vitro.

TMEV-specific T cell lines

Ag-specific T cell clones were established from the spinal cords of TMEV-infected SJL/J mice. Briefly, single-cell suspensions of spinal cords from mice perfused with PBS were prepared as described previously (22). Lymphocytes were collected from the interface of a 100/50% discontinuous Histopaque gradient (Sigma, St. Louis, MO) and then cultured with either UV-inactivated virus or peptides in the presence of irradiated syngeneic splenocytes and 10 U/ml rIL-2 (Genzyme Diagnostics, Cambridge, MA). T cell lines were maintained by biweekly stimulation with UV-inactivated virus or peptides, in the presence of 5 U/ml rIL-2.

Isolation of DC and macrophages

DC were isolated directly from splenic cell suspensions by using anti-CD11c-coated magnetic beads (MACS; Miltenyi Biotec, Auburn, CA). Such DC preparation represented >95% purity based on staining with anti-MHC class II as well as anti-CD11c (N48) Abs. Macrophages were isolated from the peritoneal cavity by flushing with 5 ml cold HBSS. Peritoneal exudate cells were then seeded on tissue culture plates, allowed to adhere for 30–60 min at 37°C, subsequently washed, and cultured in DMEM supplemented with 10% FCS.

T cell proliferation assay

Spleen or LN cells (5 x 105) were cultured in 96-well flat-bottom plates in RPMI 1640 containing 0.5% syngeneic mouse serum and 5 x 10-5 M 2-ME. Triplicate cultures were stimulated with UV-inactivated TMEV (12.5 µg/ml) for 72 h. Cultures were then pulsed with 1.0 Ci of [3H]TdR and harvested 18 h later. Measurements of [3H]TdR uptake by the cells were determined in a scintillation counter and expressed as cpm. T cell lines were similarly tested for Ag specificity (22). Briefly, 2 x 104 Histopaque-purified T cells were cultured for 72 h with the appropriate Ag, in the presence of 5 x 105 irradiated, syngeneic splenocytes without exogenous IL-2.

Ag presentation assays

T hybridoma cells (1 x 105) were cultured in triplicate in flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) for 24 h with varying concentrations of Ag or PBS in the presence of APC. T cell hybridoma stimulation was based on IL-2 production measured by the ability of the culture supernatants to support proliferation of the IL-2-dependent cell line, CTLL2. Briefly, 100 µl of supernatants were added to 7.5 x 103 CTLL2 cells in 100 µl of culture medium. After 24 h, cells were pulsed with [3H]TdR (1 µCi/well) and incubated for an additional 14–18 h before harvesting. Data represent {Delta}cpm, in which background levels of [3H]TdR uptake in cultures with PBS alone were subtracted from the levels of proliferation to Ag (the mean cpm of triplicate cultures ± SE).

Measurement of cytokine levels

Cytokine levels produced by splenic T cells in response to viral epitopes were assessed using ELISA. Briefly, nylon wool-isolated splenic T cells (2–3 x 105/well) from TMEV-infected SJL/J mice were stimulated with various concentrations of peptides or UV-TMEV for 72 h, in the presence of either 2.5 x 105 irradiated syngeneic splenocytes (3000 rad) or 5 x 104 DAS.15, I-As transfectants (32). Cell-free supernatants were examined for the presence of IL-4 or IFN-{gamma} by cytokine capture ELISA. IL-4 levels were determined using ELISA MiniKits (Endogen, Cambridge, MA). IFN-{gamma} levels were determined using capture ELISA as previously described (22). In addition, culture supernatants of isolated DC and macrophages exposed in vitro to wild-type and variant TMEV (10 PFU/cell) for 24 h were used to measure the levels of IL-12 and IL-10. IL-12p40 and IL-10 levels were measured using OPTEIA kits (BD PharMingen, San Diego, CA).

RT-PCR and RNase protection assay

Total cellular RNA from splenocytes, DC, macrophages, and neonatal astrocytes and homogenized CNS tissues was isolated by using the guanidine isothiocyanate method (33, 34) mRNA was then reverse transcribed into cDNA using oligo(dT)15–18 and murine leukemia virus reverse transcriptase. The relative concentrations of cDNA were equalized among the groups based on the level of {beta}-actin amplification (35 cycles) by PCR. Primers for control {beta}-actin and cytokines (IFN-{gamma}, IL-4, IL-10, and IL-12) were purchased from Clontech Laboratories (Palo Alto, CA). For the RNase protection assay, cellular total RNA (~10–20 µg) was hybridized using a cytokine multiprobe set (BD PharMingen) per manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of the mutation at position 244 of the VP1 capsid protein on T cell reactivity

Previously, we have demonstrated that a naturally occurring low pathogenic variant TMEV, M2, contains a single amino acid change within the entire viral coat proteins at position 244 of VP1 (GenBank accession number AF030574) (27). This mutation results in a lysine to arginine change within the predominant T cell epitope (VP1233–250) in VP1 (Fig. 1GoA). To understand the pathogenic mechanisms involved in immune-mediated demyelination, the potential effects of such a conserved amino acid substitution on T cell recognition of this region were investigated. Initially, the reactivity of a representative VP1233–250-specific Th clone (TV-3.11) derived from the infiltrates of demyelinating CNS in wild-type TMEV-infected mice (22, 23) was tested against synthetic peptides representing the VP1 epitopes of the wild-type and the variant viruses (Fig. 1GoB). This Th1 clone was strongly responsive to VP1233–250 of the wild-type virus but failed to react with the mutant peptide (VP1K244R) of the low pathogenic variant virus. The lack of reactivity to VP1K244R was not likely due to inappropriate dose of the peptide given that a wide range of peptide concentrations could not stimulate this T cell clone (Fig. 1GoB).



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  		FIGURE 1. Reactivity of T cell clones derived from TMEV-infected mice to VP1233–250 and VP1K244R. A, Schematic diagram representing a single amino acid variation in the VP1 T cell epitope region of a naturally occurring low pathogenic TMEV variant. M2 virus contains a point mutation (a->g) at nucleotide 3733 of VP1 that results in a lysine to arginine change at amino acid residue 244 of the VP1 capsid protein. The uppercase letter indicates the nucleotide substitution responsible for altering the amino acid at position 244. B, A representative VP1233–250-specific T cell clone (TV-3.11) derived from demyelinating lesions of SJL/J mice following viral infection was examined for the reactivity with a wide range of the wild-type (WT; VP1233–250) and mutant VP1 (VP1K244R) epitope peptides. Results are expressed as {Delta}cpm ± SEM ({Delta}cpm = mean cpm from stimulated cultures - mean cpm from control cultures with PBS). C, Proliferative responses to VP1233–250 and VP1K244R by T cell clones derived from mice infected with the wild-type or variant viruses were compared. T cell clones were stimulated with 10 µM concentrations of the indicated peptides in the presence of irradiated, syngeneic splenocytes for 4 days. Results are combined from several experiments and are expressed as stimulation index (mean cpm from peptide-stimulated cultures/mean cpm from hen egg lysozyme (HEL) peptide 34–45-stimulated cultures) from triplicate cultures.

 
To further examine T cell reactivity to this VP1 region in virus-infected mice, additional T cell clones derived from SJL/J mice infected with either wild-type or variant M2 viruses were tested (Fig. 1GoC). Surprisingly, a majority (9 of 13) of the VP1233–250-reactive T cell clones derived from wild-type virus-infected mice was unable to respond to the mutant peptide, VP1K244R. However, a few clones (4 of 13) were responsive to both VP1233–250 and VP1K244R. Thus, a major T cell population from wild-type virus-infected mice appears to react exclusively with the native VP1 epitope, compared with the minor population recognizing both the native and mutant forms. The possibility that M2 virus infection preferentially primes for a T cell response against the mutated VP1 T cell epitope, VP1K244R, was similarly examined with T cell clones derived from M2-infected mice (Fig. 1GoC). Conversely, the majority (five of six) of T cells derived from M2-infected mice responded to VP1K244R but not to the wild-type VP1233–250 peptide. This result suggests that M2 virus also predominantly induces a T cell response specific to the mutated VP1 epitope present in the M2 virus. Thus, substitution of an amino acid within a predominant Th epitope with a similar residue is able to preferentially induce a distinct T cell response to the respective sequences, in addition to some T cells reactive to both native and mutant epitopes. This preferential exclusive recognition of the mutant VP1 epitope by T cells from low pathogenic M2 variant virus may account for our previous observation that infection of SJL/J mice with the variant virus does not generate a strong T cell response against the wild-type VP1233–250 peptide (27).

Cytokine production by T cell clones in response to VP1233–250 and VP1K244R

To examine the possibility of a differential Th1/Th2 induction by the low pathogenic M2 virus, the production of two key Th1/Th2 cytokines (IFN-{gamma} and IL-4) by T cell clones derived from the wild-type and M2 virus-infected mice in response to the respective epitopes was assessed (Fig. 2GoA). T cell clones derived from wild-type TMEV-infected mice produced predominantly IFN-{gamma} on stimulation with VP1233–250. The majority (five of seven) of the T cell clones produced high levels of IFN-{gamma} with minimal levels of IL-4. This is consistent with the previous studies demonstrating that a Th1 response is predominantly induced against this epitope following infection with pathogenic TMEV (23). Interestingly, the majority (four of five) of M2-derived T cell clones preferentially produced IL-4 after stimulation with the variant peptide, VP1K244R, suggesting a prominent Th2 response. Only one clone produced a relatively high level of IFN-{gamma}, although a low level of IL-4 production was also detected. The T cell clones producing both cytokines may represent a Th0 population, yet to be differentiated further to either Th1 or Th2. These data strongly support the possibility that infection with the low pathogenic M2 virus results in the preferential generation of noninflammatory Th2 cells, in contrast to the Th1 response induced following the wild-type virus infection.



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  		FIGURE 2. Activation and cytokine production by T cell clones derived from wild-type or variant virus-infected mice in the presence of wild-type and mutant VP1 epitopes. A, IFN-{gamma} and IL-4 production by wild-type virus and M2-derived T cell clones in response to VP1 epitope peptides. Various T cell clones derived from the wild-type virus-infected mice were stimulated with VP1233–250 and M2-derived T cell clones were stimulated with VP1K244R. Culture supernatants were collected after ~70–72 h and assayed for the production of IFN-{gamma} and IL-4 by capture ELISA, as described in Materials and Methods. IFN-{gamma} and IL-4 production in response to a nonspecific control peptide, HEL34–45, was <2 ng/ml and 1 pg/ml, respectively, for the T cell clones analyzed. B, Selected T cell clones derived from either SJL/J mice infected with the wild-type (WT) or variant (M2) virus, were assessed for proliferation and induction of IFN-{gamma} and/or IL-4 in response to the respective VP1 epitope peptides. The levels of cytokines were determined by ELISA.

 
It has previously been shown that certain altered peptide ligands can stimulate IL-4 production by Th2-type clones and cytolysis by Th1-type clones, without inducing a significant level of proliferative response (35, 36). To determine whether the mutant VP1K244R peptide is capable of inducing cytokine production without proliferation in VP1233–250-specific T cell clones and vice versa for the wild-type peptide in VP1K244R-specific clones, both proliferation and cytokine production by selected T cell clones in response to the epitope peptides were assessed (Fig. 2GoB). The ability of the peptides to induce proliferative responses correlated with the stimulation of IFN-{gamma} or IL-4 production, i.e., clones that did not proliferate also did not produce the cytokines. In addition, the proliferative response corresponded well with the peptide-dependent cytolysis by Th1-type clones (data not shown). However, there was again no correlation between the Th type and the dual epitope recognition (VP1233–250 and VP1K244R), indicating that the differences in epitope recognition may not be a crucial factor in determining the Th type.

Epitope specificity and type of Th cells induced in mice following viral infection

To investigate the bulk T cell levels specific for the VP1 (VP1233–250 and VP1K244R) and other major epitopes in mice infected with either wild-type or variant TMEV, T cell-proliferative responses to the major T cell epitopes were examined at 30 day post-viral infection (Fig. 3Go). The level of T cells specific for the VP1 epitope from the wild-type virus-infected mice were similar to the level of T cells reactive to the respective (mutant) VP1 epitope in the variant virus-infected mice at the onset of the disease. In addition, very low proliferative response was seen against the mutant VP1 epitope in wild-type virus-infected mice and vice versa, suggesting that only a minor population of T cells recognize both VP1 epitopes in mice infected with wild-type or mutant virus (data not shown and Ref. 27). The levels of VP2- as well as VP3-reactive T cells were very similar at this time point in both the wild-type and the variant virus-infected mice. To examine the possibility that low pathogenic virus infection preferentially induces Th2 response, as opposed to the predominant Th1 response by the pathogenic wild-type virus, the production of IL-4 and IFN-{gamma} by bulk splenic T cell cultures from M2 virus-infected mice were compared with that from the wild-type virus-infected mice (Fig. 3Go). Interestingly, high levels of IL-4 were produced by T cells from M2-infected mice in response to VP1K244R, as well as to the other predominant T cell epitopes, VP274–86 and VP324–37. In particular, the level of IL-4 produced in response to the VP1 region was significantly greater in M2-infected mice than in wild-type virus-infected mice (p = 0.03). In contrast, the wild-type virus-infected mice produced a relatively low level of IL-4 in response to VP1233–250 or VP274–86 (except to VP324–37), whereas a significantly higher level of IFN-{gamma} was produced in response to VP1233–250 than in M2-infected mice. Overall, higher levels of Th1 than of Th2 cytokines were also produced in response to wild-type TMEV, and the opposite profile was seen in response to the variant virus (data not shown). These results indicate that the variant virus, consistent with the low pathogenicity of demyelinating disease, induces relatively higher levels of the Th2 response to viral epitopes.



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  		FIGURE 3. Proliferative responses and cytokine production by bulk T cells from wild-type and variant virus-infected mice in response to the predominant T cell epitopes. Splenocytes of intracerebrally infected SJL/J mice (three per group) at day 30 postinfection with either the wild-type or variant virus (3 x 106 PFU) were assessed for the specific proliferative responses to the predominant T cell epitopes, including VP1233–250 and VP1K244R. The proliferative responses were determined as described in Materials and Methods. The production of IL-4 and IFN-{gamma} by splenic cultures from SJL/J mice infected with wild-type or variant (M2) virus in response to the wild-type or variant VP1 peptide was assessed. Splenic T cells were isolated on nylon wool columns and subsequently cultured (3 x 105/well) with 1 µM concentrations of the indicated peptides in the presence of 5 x 104/well DAS.15 I-As fibroblast transfectants as a source of APC. Culture supernatants were collected after 72 h and assayed for the presence of IL-4 and IFN-{gamma} by capture ELISA. Results are expressed as picograms per milliliter ± SD of IL-4 or IFN-{gamma}. The level of IL-4 produced in response to the VP1 region was significantly greater in M2-infected mice than in wild-type virus-infected mice (p = 0.03).

 
Th cell types induced following immunization with VP1 epitope peptides or TMEV

To determine whether VP1K244R peptide with the amino acid substitution (K->R) could intrinsically affect the stimulation of Th-type cells in vivo, SJL/J mice were immunized with VP1233–250 or VP1K244R. The recall proliferative responses as well as cytokine production by LN cells in response to the peptides were then measured (Fig. 4Go). Immunization with VP1233–250 generated a strong proliferative response to VP1233–250 and a significantly reduced response to VP1K244R; 89.1% reduction (p < 0.0001) at 0.1 µM peptide concentrations (Fig. 4GoA). Conversely, immunization with VP1K244R peptide generated a strong proliferative response to VP1K244R, but a much lower proliferative response to the wild-type VP1233–250 peptide; 68.9% reduction (p = 0.01) at 0.1 µM peptide concentrations (Fig. 4GoA). Thus, despite the conservative amino acid substitution, the induction of T cell populations cross-reactive for this VP1 region appears to be greatly affected by this change following immunization, as seen with T cells from virus-infected mice. However, RT-PCR assessment for cytokines indicates that these cells activated by the respective peptides induce IFN-{gamma} as well as IL-4 messages, in contrast to the differential Th type responses found in virus-infected mice (Fig. 4GoB). Similarly, both VP1233–250 and VP1K244R were able to induce strong IFN-{gamma} production (Fig. 4GoC). These results indicate that direct immunization with the wild-type as well as the variant VP1 epitopes induced both Th1 and Th2 responses without apparent predominance of one particular Th type.



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  		FIGURE 4. Comparison of T cell responses in mice immunized with VP1233–250 or VP1K244R peptide. SJL/J mice (three mice per group) were immunized with epitope peptides within the VP1 protein of the wild-type (VP1 or VP1-W) or M2 virus (K244R or VP1-M) emulsified in CFA. Mice were also immunized with PBS emulsified in CFA (PBS/CFA), as a negative control. Ten days later, LN cells were pooled and subsequently cultured with various molar concentrations of VP1233–250 or VP1K244R for 4 days. The positive control proliferative responses to a mycobacterial lysate containing PPD were comparable: PBS/CFA = 76, 987 cpm; VP1233–250/CFA = 72,870 cpm; VP1K244R/CFA = 71,962 cpm. A, The proliferation results are expressed as the {Delta}cpm ± SEM (mean cpm of peptide-stimulated cultures - mean cpm of control HEL34–45-stimulated cultures). B, The levels of cytokine mRNAs of IFN-{gamma} and IL-4 in the cell pellets were determined by RT-PCR as described in Materials and Methods. C, IFN-{gamma} levels in the supernatants of replicate cultures with 1 µM Ag concentration were determined by ELISA. VP1233–250 response in VP1233–350-immunized mice, p < 0.0001; VP1K244R response in VP1K244R-immunized mice, p = 0.01.

 
To address the possibility that the above nonskewed Th1/Th2 responses to the epitope peptides following peptide-immunization may not represent T cell responses to the whole virions due to the potential differences in the Ag processing, UV-inactivated virus was used to similarly immunize SJL/J mice (Fig. 5Go). The levels of proliferation and IFN-{gamma} induction were again very similar between mice immunized with wild-type and low pathogenic variant viruses. These results strongly suggest that the differential induction of Th1/Th2 responses following infection with these viruses is not due to differences in the Ag processing and presentation. Therefore, the intrinsic difference in this epitope region is not likely responsible for the Th2 skewing in mice infected with the variant virus.



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  		FIGURE 5. T cell responses in mice immunized with UV-inactivated wild-type or mutant TMEV. SJL/J mice (three to four mice per group) were immunized with UV-inactivated wild-type (WT) or mutant (M2) virus emulsified in CFA similar to experiments in Fig. 4Go. Mice were also immunized with HEL emulsified in CFA as a negative control. A. Proliferation of LN cells from mice immunized with purified UV-TMEV (wild-type or M2) in response to the immunogens. Data are represented as {Delta}cpm ± SEM (cpm of UV-TMEV-stimulated cultures - cpm of PBS-stimulated cultures). B, IFN-{gamma} and IL-4 mRNA in the LN cells from immunized mice were similarly assessed by RT-PCR after restimulation with corresponding UV-TMEV (wild-type or variant M2).

 
Differential induction of IL-12/IL-10 by the wild-type and variant viruses in APCs

Because the viral peptides themselves do not intrinsically induce differential Th type responses, it is conceivable that APC involved in the initial T cell activation may respond differentially to the pathogenic and nonpathogenic variant viruses. To examine this possibility, isolated APC populations (DC and macrophages) were exposed to the viruses, and then the induction of key cytokines (IL-10 and IL-12) known to influence the development of Th type responses was assessed (Fig. 6Go). Interestingly, DC exposed to the wild-type virus preferentially induced IL-12 gene expression with a reduced level of IL-10 transcription, whereas those exposed to the variant virus exhibited a higher level of IL-10 transcripts without elevated IL-12 gene expression. Similarly, peritoneal macrophages preferentially induced IL-12 gene transcription following exposure to the wild-type virus and IL-10 to the variant virus (Fig. 6GoA), although the induction kinetics was slower (24 h) than that in DC (5 h). Further analyses of culture supernatants of virus-infected APC by cytokine-specific ELISA concur with the cytokine messages detected (Fig. 6GoB). For example, >5-fold in IL-12 production was induced by wild-type virus compared with the low pathogenic variant virus, and an inverse increase in IL-10 production was induced in DC cultures by the variant virus at 24 h postinfection. In addition, virus-infected DC as well as macrophages specifically stimulated virus-specific T cell hybridoma cells in vitro, clearly indicating the possibility that virus-infected APC can also stimulate virus-specific class II-restricted Th cells (data not shown). Because IFN-{gamma}-activated primary astrocytes are able to present viral Ags to T cells and may play a role in the pathogenesis of immune-mediated demyelination (34), the possibility of similar induction of proinflammatory cytokines by TMEV infection in astrocyte cultures was assessed (Fig. 6GoC). Infection of astrocytes with wild-type virus strongly induced IL-12 and IL-1 compared with the mutant M2 virus. This suggests that the pathogenic wild-type TMEV is capable of inducing higher levels of proinflammatory cytokines than low pathogenic variant virus even in the nonprofessional APC. TMEV inactivated by UV irradiation was unable to induce such cytokines (IL-12 or IL-10) in DC (Fig. 7Go), similar to chemokine gene activation in astrocyte cultures (37), suggesting that productive viral infection is necessary for the cytokine/chemokine gene activation. Overall, these results are consistent with our observations that the Th1-type response is preferentially induced in the wild-type virus-infected mice and the Th2 response in mice infected with the low pathogenic variant virus.



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  		FIGURE 6. Differential induction of IL-12/IL-10 in APC after exposure to wild-type (WT) and variant viruses. A, Isolated DC and macrophages were exposed to the wild-type or the variant virus (10 PFU/cell) for 5 and 24 h. The control (Con) uninfected and virus-infected cultures were then subjected to RT-PCR for assessment of IL-12 and IL-10 cytokine message levels. B, Culture supernatants from isolated DC and macrophages exposed to TMEV for 24 h were assessed for the presence of IL-12 and IL-10 proteins by ELISA. The experiments were repeated several times, and a representative result is shown here. C, The induction of IL-12 and IL-1 was assessed by RNase protection assay in primary astrocytes (nonprofessional APC) infected with TMEV for 6 h. Significantly higher levels of these cytokines were seen with the wild-type virus than with the low pathogenic M2 virus at the same multiplicity of infection, demonstrating a preferential promotion of the Th1 response by wild-type virus-infected astrocytes similar to those of DC and macrophages.

 


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  		FIGURE 7. UV inactivation of TMEV abrogates cytokine induction in DC. Cytokine induction in DC infected with intact wild-type (WT) virus was compared with that of UV-inactivated virus at the same multiplicity of infection as assessed by RT-PCR. Productive viral infection is necessary for the induction of cytokines in DC. Con, Uninfected control.

 
To test the possibility that the wild-type and mutant VP1 epitopes may be capable of differentially inducing IL-12/IL-10 cytokines in APC, DC and macrophages were incubated with the peptides (VP1233–250 and VP1K244RI) for 5 or 24 h and then the levels of cytokine messages were assessed (data not shown). These results indicate that the APC are not stimulated by any of the peptides to produce such cytokines, confirming the notion that viable virus infection is required and the peptide alone is not sufficient for differential induction of cytokines. These are consistent with in vivo experiments showing similar Th1/Th2 responses following peptide immunization (Figs. 4Go and 5Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we have isolated a spontaneously arising variant of TMEV of which the pathogenicity and persistence in the CNS were significantly lower than the those of pathogenic parent virus (27). However, an intracerebral inoculation of this variant virus following inflammatory responses to bacterial LPS induced a similar level of viral persistence and demyelinating disease (38). The overall T cell proliferative responses to the respective viruses in mice infected with parent or variant viruses were not significantly different from each other. However, VP1233–250-specific T cell populations induced by the parent virus poorly responded to the variant virus. Further analyses indicated that there was a single substitution of lysine to arginine at position 244 of a major Th epitope, VP1233–250 (GenBank accession number AF030574), within the entire P1 region encoding all the viral capsid proteins (27). The substitution at VP1244 appears to be responsible for the low pathogenicity because this residue is the only one of two residue substitutions (another change in a putative viral protease, 2A) in the entire viral genome consistently different from the wild-type virus in several similarly obtained low pathogenic viruses (J.-A. Kang and B. S. Kim, unpublished observation).

In this study, predominance of the Th type response induced by a low pathogenic variant TMEV was analyzed to correlate with the pathogenicity. Our results indicate that the pathogenic virus induces predominantly the Th1 type, whereas the low pathogenic variant virus induces the Th2-type response (Figs. 2Go and 3Go). To our knowledge, this is the first example that such substitution within a Th epitope of a spontaneously occurring variant virus significantly alters the viral pathogenicity by inducing an alternate Th-type response. Previously, several investigators have also demonstrated that spontaneously occurring variants of different viruses contain similar substitutions within the major CTL epitopes and that virus-infected cells are able to escape from CTL-mediated lysis (39, 40, 41). Thus, such an amino acid substitution within the major T cell epitope for Th cells as well as CTL may provide an important means for the virus to evade the immune system (27, 42). Furthermore, many diseases induced by various microorganisms or immunization with autoantigens display skewed Th1 or Th2 responses toward the organism and/or autoantigens (43, 44, 45, 46). Consequently, alterations in the predominance of the Th-type response may lead to protection from TMEV-induced demyelinating disease, as observed after manipulating proinflammatory cytokine levels (7). Our previous study indicating that neutralization of IL-12 suppresses the severity and onset of demyelinating disease (25) is consistent with this possibility. However, there is a conflicting report regarding the effects of treatment with neutralizing Abs to IL-12 on TMEV-induced demyelination (25, 47), which may reflect the differences in the TMEV strains and time of treatment with respect to viral infection.

The mechanisms involved in the preferential induction of Th1 response by the wild-type virus compared with the variant virus are not yet clear. It is conceivable that VP1233–250 may have a critical role in the initial establishment of the preferential Th-type-specific response and that the altered VP1 epitope of the variant virus may provide a microenvironment favoring a Th2 response. Consequently, the lack of initial predominance of Th1 response following the variant virus infection may result in its low pathogenicity. We have previously demonstrated that the preferential induction of Th1 responses to major viral epitopes (i.e., Th1 for VP1233–250 and VP274–86 vs Th2 for VP324–37) following viral infection is associated with the pathogenicity of demyelinating disease (23). Because the above three Th epitopes represent the majority (>85%) of Th responses in SJL/J mice (26), this amino acid substitution in the VP1 epitope (27) may be able to alter the balance in favor of nonpathogenic Th2 responses to the variant virus (Fig. 3Go). In addition, detection of Th1 cytokines before Th2 cytokines in the CNS of susceptible mice infected with TMEV suggests that the initial establishment of the Th-type response is likely important for sustaining pathogenic, inflammatory Th1 responses (38). Furthermore, Falcone and Bloom (48) recently demonstrated that the initial microenvironment favoring a Th2-type response by coimmunization with an unrelated Ag prevents the induction of EAE. This is consistent with the observation that the variant virus preferentially induce a Th2 response in mice even to an unaltered VP274–86 epitope (Fig. 3Go), which normally induces a Th1 response in the wild-type virus-infected mice (23). Moreover, the fact that exposure to the low pathogenic variant virus before the wild-type virus infection renders strong protection from virally induced demyelination (23) supports this possibility. The early establishment of predominant Th2 responses to viral epitopes on pre-exposure to the variant virus may contribute to the protection. However, direct immunization with the epitope peptides or UV-inactivated virus could not duplicate the preferential Th-type responses following viral infection (Figs. 4Go and 5Go), strongly suggesting that other mechanisms such as differences in the initial response to viral infection by APCs may be involved.

Our experimental results indicate that professional APCs (e.g., DC and macrophages) respond differently to these viruses. Following viral exposure in vitro, the wild-type virus is apparently capable of preferentially inducing IL-12, promoting a Th1 response, and the low pathogenic variant virus IL-10, promoting a Th2 response (Figs. 6Go and 8Go). However, UV inactivation of TMEV abolished cytokine induction, indicating the requirement of intact virus (Fig. 7Go), similar to that seen in the abrogation of chemokine induction in astrocyte cultures (37). It has recently been shown that infection with other viruses such as dengue virus or parainfluenza virus can activate DC to produce IL-12 (49, 50). However, our low pathogenic variant virus can preferentially induce IL-10 over IL-12. Previously, it has also been reported that a low dose of influenza virus preferentially induces IL-12 and a high dose IL-10 in DC (51). Our observation is apparently the first report indicating that the same dose of virus infection induces differential IL-12/IL-10 production in professional APC, depending on the viral pathogenicity, and this may represent an important underlying mechanism for the differential pathogenesis of variant viruses in inflammatory diseases. The induction of IL-12 by the wild-type TMEV is not limited to professional APC, given that this virus induces such proinflammatory cytokines much more efficiently than the mutant M2 virus (Fig. 6GoC). These results strongly suggest that nonprofessional APC, such as astrocytes in the CNS may contribute to the preferential induction of pathogenic Th1 responses by the wild-type TMEV. This early establishment of the microenvironment following viral infection may be able to determine the Th-type response and consequently the outcome of pathogenicity of the viruses. A similar initial production of proinflammatory cytokines may also occur in vivo after viral infection, because certain viral infections lead to a high level of IL-12 induction in virus-infected mice (52, 53, 54, 55). Unrelated inflammatory responses of the host apparently enhance viral persistence of the wild-type virus in resistant C57BL/6 mice (56) and this variant virus in susceptible SJL mice (38). Thus, the initial inflammatory Th1 response following wild-type virus infection may also increase viral persistence, whereas the lack of initial inflammatory Th1 response following variant virus infection may not be able to promote viral persistence (27).



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  		FIGURE 8. Schematic representation of a potential mechanism for immune-mediated demyelination by wild-type compared with the variant TMEV. Greater levels of Th1 response than of Th2 response to viral epitopes are generated in the wild-type virus-infected mice leading to pathogenic demyelination. In contrast, higher levels of Th2 response than Th1 response to viral epitopes are generated in the low pathogenic variant virus-infected mice. A single amino acid substitution within the pathogenic VP1 epitope in the variant virus appears to influence the predominance of a Th2 response in the virus-infected mice via differential IL-12/IL-10 induction in the APC that are initially exposed to the viruses. Higher levels of IL-12 induced by the wild-type virus may be able to skew toward Th1 responses to viral epitopes, leading to pathogenesis of demyelination. In contrast, the preferential induction of IL-10 by variant virus-infected APC may favor Th2 responses to viral epitopes that are not pathogenic.

 
The molecular mechanisms involved in the differential cytokine responses by the APC are not yet known, and further studies will be necessary to define the mechanisms. Our preliminary in situ hybridization results for viral RNA strongly suggest that initial viral replication levels in the CNS of infected mice as well as in isolated DC are indistinguishable between the wild-type and variant viruses (data not shown). However, it is possible that the wild-type and variant viruses may preferentially infect different subpopulations of DC that are programmed to produce IL-12 or IL-10, respectively (57, 58). The differences in the VP1 sequences may allow preferential virus infection to such subpopulations of APC, perhaps at different stages of differentiation and/or origin. The fact that the position (VP1244) of the amino acid change is in close proximity to the strongly neutralizing, putative receptor-binding site (VP1265–276) supports this possibility (59). Induction of IL-12 production was also reported following various bacterial infections via engagement of Toll-like receptors by bacterial LPS or lipoproteins (60, 61). However, it is not yet known how TMEV infection triggers IL-12 production, and further studies would be necessary to determine the mechanisms. Alternatively, the viral protein altered in the variant virus may be directly involved in the different transcriptional regulation of these cytokine genes. Furthermore, the wild-type virus may induce necrosis and the variant virus apoptosis in a subpopulation of infected APC, such as DC (62). This in turn differentially promotes the maturation of DC and activation of inflammatory Th1 cells as well as perhaps production of IL-12 following wild-type virus-infection, in contrast to variant virus infection (63, 64). In an attempt to recreate the type of T cell responses following viral infection with virus-infected APC in vivo, DC infected with either the wild-type or mutant viruses in vitro were administered i.v. into naive SJL mice. However, differential Th1/Th2 response in the recipient mice was not apparent due to the high background IFN-{gamma} levels (data not shown). This might reflect the continuous viral infection of DC and/or nonspecific stimulation of T cells by the transferred, infected DC. Nevertheless, such preferential IL-12/IL-10 production will most likely lead to skewed development of Th types that are subsequently triggered to produce various inflammatory or noninflammatory cytokines influencing disease induction/progression (Fig. 8Go). It is particularly interesting that a spontaneously occurring variant virus induces an altered pattern of Th type response at the APC level in the initial stage of immune response leading to attenuated pathogenesis of inflammatory demyelination. Thus, understanding the mechanisms involved in such differential activation of critical cytokines by alterations in viral sequences may lead to the elucidation of pathogenic mechanisms of virally induced immune-mediated inflammatory diseases as well as provide a powerful means to control viral diseases.


   Footnotes
 
1 This work was supported by the U.S. Public Health Service (Grants NS 28752 and NS 33008), the National Multiple Sclerosis Society (Grant RG2956-A3), and Postdoctoral Fellowship FG1172-A-1 from the National Multiple Sclerosis Society (to J.P.P.). Back

2 J.P.P. and R.L.Y. contributed equally to this work. Back

3 Current address: Sugen, Inc., South San Francisco, CA 94080. Back

4 Current address: Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115 Yusong, Taejon, Korea. Back

5 Address correspondence and reprint requests to Dr. Byung S. Kim, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: bskim{at}northwestern.edu Back

6 Abbreviations used in this paper: TMEV, Theiler’s murine encephalomyelitis virus; EAE, experimental autoimmune encephalomyelitis; DC, dendritic cell; LN, lymph node; HEL, hen egg lysozyme. Back

Received for publication July 19, 2001. Accepted for publication February 15, 2002.


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 Materials and Methods
 Results
 Discussion
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J. P. Palma, D. Kwon, N. A. Clipstone, and B. S. Kim
Infection with Theiler's Murine Encephalomyelitis Virus Directly Induces Proinflammatory Cytokines in Primary Astrocytes via NF-{kappa}B Activation: Potential Role for the Initiation of Demyelinating Disease
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