Membrane lymphotoxin is required for resistance to Theiler’s virus infection

Xiaoqi Lin1, Xiaoxing Ma1, Moses Rodriguez3, Xuan Feng1, Laurie Zoecklein3, Yang-Xin Fu2 and Raymond P. Roos1

Departments of 1 Neurology and 2 Pathology, University of Chicago, Chicago, IL 60637, USA and 3 Departments of Neurology and Immunology, Mayo Clinic, Rochester, MN 55905, USA

Correspondence to: Y.-X. Fu; E-mail: yfu{at}midway.uchicago.edu and R. P. Roos; E-mail: rroos{at}neurology.bsd.uchicago.edu.
Transmitting editor: L. Steinman


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphotoxin (LT) and tumor necrosis factor (TNF) are important in immune system development and function. LT consists of soluble LT-{alpha}3, which binds to TNF-R1 and TNF-R2, and membrane LT-{alpha}1ß2, which binds to LT-ß-R. We investigated the role of LT and TNF in disease induced by Daniel’s (DA) strain of Theiler’s murine encephalomyelitis virus (TMEV) since the immune response is believed to be important in both resistance to DA infection as well as mediation of virus-induced demyelination. DA persisted and induced inflammatory demyelination in LT-{alpha}–/– (but not TNF–/–) weanling mice of a normally resistant haplotype (C57BL/6), suggesting that LT, but not TNF, is critical for resistance to DA infection. This activity of LT depends on membrane LT-{alpha}1ß2 and not soluble LT-{alpha}3, since DA virus persisted and induced inflammatory demyelination in LT-ß-R–/–, but not TNF-R1–/– or TNF-R2–/–, mice. The LT-{alpha}–/– and LT-ß-R–/– mice failed to mount a virus-specific cytotoxic T cell response. Treatment of weanling C57BL/6 mice with LT-ß-R–Ig, which blocks membrane LT activity, failed to increase susceptibility, suggesting that the LT effect is related to its action on immune system development which is fixed by 3 weeks of age. Our data suggest that membrane LT is important in resistance to DA infection (possibly through interference with CD8+ T cell development and function). There was relatively little demyelination associated with inflammation in LT-{alpha}–/– and LT-ß-R–/– mice compared to susceptible SJL mice, suggesting the possibility that LT plays a role in mediating demyelination.

Keywords: cytokine, cytotoxic T lymphocyte, demyelination, lymphotoxin, Theiler’s virus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Daniel’s (DA) strain of Theiler’s murine encephalomyelitis virus (TMEV) is cleared from the central nervous system (CNS) of resistant strains of mice (H-2b,k), but induces a biphasic disease in the CNS of susceptible strains (H-2s,q,v) characterized by an acute encephalitis followed by a persistent infection with chronic inflammatory demyelination (1,2). DA-induced disease serves as a model system for the study of multiple sclerosis because of the similarity in the demyelinating disease pathology and because the immune system appears to contribute to the white matter disease in both. The immune system appears to play a dual role, decreasing the amount of virus early after infection, but fostering demyelination late (3). The mechanisms by which mice are resistant/susceptible to this potentially lytic virus and develop demyelination from a persistent TMEV infection are not completely understood, but appear to involve both class I-restricted CD8+ T cells and class II-restricted CD4+ T cells (28), and cytokines (912). The effect of lymphotoxin (LT) and tumor necrosis factor (TNF) on TMEV disease is not clearly defined (1316); however, these factors are known to have an influence on other virus infections (1720).

LT, which is primarily expressed in activated T, B and NK cells (21,22), is present in two forms, soluble LT-{alpha}3 homotrimers and membrane-associated LT-{alpha}1ß2 heterotrimers. Soluble LT-{alpha}3 is structurally related to TNF-{alpha}3 and binds either of two TNF receptors, TNF-R1 and TNF-R2. Soluble LT-{alpha}3 and TNF-{alpha}3 induce similar effects by activating a wide variety of inflammatory and immune responses. In contrast, membrane LT-{alpha}1ß2 heterotrimers have high-affinity binding to the LT-ß receptor (LT-ß-R), without binding to TNF-R1 and R2.

LT and TNF have an important role in the development of the immune system, the differentiation of immune progenitor cells, and in the activation of transcription factors which regulate the inflammatory response and death and survival pathways (21,22). LT-{alpha}–/– (which are deficient in both soluble LT-{alpha}3 and membrane LT-{alpha}1ß2), LT-ß–/– (which are deficient in membrane-associated LT-{alpha}1ß2) and LT-ß-R–/– mice have profoundly defective development of the peripheral lymphoid organs (21). LT-{alpha}–/– mice and mice in which membrane LT-{alpha}1ß2 has been blocked by an LT-ß-R–Ig fusion protein have a markedly reduced number of dendritic cells (DC) in the spleen as well as a decreased contact sensitivity and primary IgG response (2325).

In this report, we characterized the role of LT and TNF in TMEV disease. We found that membrane LT-{alpha}1ß2, but not soluble LT-{alpha}3 and TNF, plays an important role in determining resistance to DA infection and DA-induced disease. The data suggested that a deficiency of membrane LT led to abnormal development and function of anti-virus CD8+ cytotoxic T lymphocytes (CTL), preventing efficient clearance of TMEV in a normally resistant mouse strain; the lack of the anti-virus CTL response led to virus persistence with the subsequent induction of white matter disease. Interestingly, data also raised the possibility that LT-{alpha}1ß2 was not only important in mediating resistance to TMEV infection, but also in fostering the demyelinating pathology.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Virus
Wild-type DA strain of TMEV derived from an infectious clone pDAFL3 was plaque-purified, propagated in BHK-21 cells and titered on monolayers of L-2 cells as previously described (10).

Animals
Female C57BL/6 mice (4–6 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME); 4- to 6-week-old female C57BL/6 LT-{alpha}–/– mice deficient in both soluble LT-{alpha}3 and membrane LT-{alpha}1ß2, C57BL/6 LT-ß-R–/– mice deficient in the receptor for membrane LT-{alpha}1ß2, C57BL/6 TNF–/– mice deficient in TNF, C57BL/6 TNF-R1–/– mice deficient in TNF-R1, and C57BL/6 TNF-R2–/– mice deficient in TNF-R2 (25) (Jackson Laboratory) were bred in the University of Chicago animal facility under specific pathogen-free conditions. Mouse care and use were in accordance with institutional guidelines. Weanling mice were intracerebrally inoculated with 6 x 105 p.f.u. of DA virus.

Reagents
Protein A column-purified LT-ß-R–Ig fusion protein, which binds LT-{alpha}1ß2 and blocks the effect of membrane LT-{alpha}1ß2, was prepared as previously described (25). Selected mice received i.p. inoculation(s) of 100 µg LT-ß-R–Ig fusion protein in a volume of 100 µl with two different schedules: either once at the time of virus inoculation or for a total of 3 times, starting on the day of virus inoculation and continuing at intervals of 1 week. These protocols are standard ones in our laboratory, and consistently inhibit the formation of follicular DC and germinal centers (21). A biotinylated mAb directed against DA VP1 (DAmAb1) was used for immunohistochemical identification of virus antigen (26). Alkaline phosphatase-conjugated affiniPure F(ab)2 fragment goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch, West Grove, PA) was used in an ELISA for detection of virus-specific antibody in the sera of infected mice (10).

Immunohistochemistry
The spinal cord and brainstem were embedded in paraffin, sectioned, deparaffinized and stained with biotinylated mAb against DAmAb1 according to a commercial protocol (TSA Biotin System, NEL700; NEN Life Science Products, Boston, MA).

Pathologic analysis
Mice were anaesthetized and perfused 42 days post-infection (p.i.), and spinal cords were processed for pathologic analysis as described previously (10). The total score was expressed as the percentage of spinal cord quadrants with the specific abnormality such that a maximum score of 100 represents the presence of pathology in every quadrant of every spinal cord section examined (10). Analysis of variance (ANOVA and Bonfarroni adjustment t-tests) was used to evaluate significant differences in pathological scores between different strains of mice or mice with different treatments.

NK cell activity assay
CNS-infiltrating mononuclear cells (CNS-IMNC) were isolated 7 days p.i. and used as effectors in a cytotoxicity assay with 51Cr-labeled Yac-1 cells as targets (27). NK cells activity was calculated by the average percentage of 51Cr release: [(experimental counts – spontaneous counts)/(maximum counts – spontaneous counts)] x 100% (from triplicate wells). Statistical comparisons were performed by the unpaired Student’s t-test.

Virus-specific antibody determination
Mouse sera were collected 42 days p.i. for determination of virus-specific antibody by ELISA (10).

Cytotoxicity assay
CNS-IMNC were isolated 7 days p.i as effectors, and C57SV (Kb, Db) and C57SV/LP (which expresses DA leader, VP4, VP2 and VP3) (27) were labeled with 51Cr and used as targets in a DA virus-specific cytotoxicity assay (27). Mean radioactivity values were calculated from triplicate wells and results expressed as percent-specific lysis according to the formula: (experimental counts – spontaneous counts)/(maximum counts – spontaneous counts)] x 100%. The SEM was determined from results obtained from pooled CNS-IMNC samples in triplicate wells. Statistical comparisons were performed by the unpaired Student’s t-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LT, but not TNF, determines resistance to DA virus infection in mice with a normally resistant haplotype
The DA strain of TMEV causes a persistent infection and demyelination in susceptible (e.g. SJL) but not resistant (e.g. C57BL/6) mouse strains. Following DA infection, SJL mice develop an initial subclinical gray matter disease that is replaced in 3–6 weeks by inflammatory demyelination of the white matter; there is evidence of persistent virus for the life of the mouse. In contrast, C57BL/6 mice clear virus within 2–3 weeks. Although the mechanisms of resistance versus susceptibility are not well understood, an anti-virus CTL response is believed to mediate resistance and virus clearance in C57BL/6 mice, while a failure in the CTL response is believed to foster the persistent virus infection and demyelination seen in SJL mice (37,28).

In order to clarify the role of LT and TNF in DA-induced disease, we examined the effect of infection of resistant C57BL/6 mice that were deficient in LT-{alpha} (both soluble LT-{alpha}3 and membrane LT-{alpha}1ß2) and TNF. Following DA infection, three of seven C57BL/6 LT-{alpha}–/– mice developed complete paralysis and died by 4 weeks p.i., while two had hind limb paralysis at the time of sacrifice 6 weeks p.i. In contrast, no clinical deficits were observed following inoculation of wild-type C57BL/6 and C57BL/6 TNF–/– mice. SJL mice failed to develop clinical signs 6 weeks p.i., although six of 20 became paralyzed 4 months p.i. These results indicate that DA infection causes clinical deficits in a normally resistant mouse strain deficient in LT-{alpha}, but not TNF. The data suggest that LT-{alpha}, but not TNF, plays a role in resistance to DA infection in C57BL/6 mice.

To test whether DA virus produced a persistent infection in C57BL/6 LT-{alpha}–/– mice, we examined the spinal cord of mice 42 days p.i. for DA virus antigen by immunohistochemical staining with biotinylated DAmAb1. DA virus antigen was detected in the spinal cord of C57BL/6 LT-{alpha}–/– mice, but not TNF–/– mice or wild-type C57BL/6 (data not shown). Therefore, LT-{alpha}, but not TNF, appears critical for DA virus clearance from the CNS.

LT may play a role in mediating demyelination
To test whether infected C57BL/6 LT-{alpha}–/– mice develop CNS inflammation and demyelination similar to that seen in SJL mice, we examined spinal cord sections 42 days p.i. As expected, SJL mice developed prominent inflammation as well as demyelination in the white matter (Fig. 1H and Table 1), usually with the presence of inflammatory cells within the demyelinated lesions. In contrast, C57BL/6 LT-{alpha}–/– mice developed numerous inflammatory infiltrates in the white matter with only minimal evidence of demyelination 42 days p.i. (Fig. 1B and Table 1), i.e. although there was approximately half as much inflammation in lesions in C57BL/6 LT-{alpha}–/– compared to SJL mice, the lesions in C57BL/6 LT-{alpha}–/– generally showed far less demyelination than that seen with SJL (Fig. 2B and D, and Table 1). Although these comparisons are between different mouse strains, the results suggest that the LT-regulated immune response may play a role in the development of demyelinating lesions induced by DA infection. Few, if any, lesions were observed 42 days p.i. in wild-type or TNF–/– mice (Fig. 1A and C, and Table 1), presumably because virus was cleared in these mice.



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Fig. 1. Histopathological findings in the spinal cord of DA virus-infected mice. Mice were perfused 42 days p.i., and the spinal cord was collected, embedded, sectioned and stained with a modified erichrome method with crystal violet counterstain. Inflammation and demyelination were observed in the spinal cord of C57BL/6 LT-{alpha}–/– (B), C57BL/6 LT-ß-R–/– (F) and SJL (H) mice, but not in wild-type C57BL/6 (A), C57BL/6 TNF–/– (C), C57BL/6 TNF-R1–/– (D) and C57BL/6 TNF-R2–/– mice (E) or C57BL/6 mice treated with LT-ß-R–Ig (G).

 

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Table 1. Pathological changes in the spinal cords of mice 42 days p.i. with DA virus
 


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Fig. 2. Inflammation and demyelination in the spinal cord of DA virus-infected mice. Mice were perfused 42 days p.i., and the spinal cord was collected, embedded, sectioned and stained with a modified erichrome method with crystal violet counterstain. Foci with inflammatory cells but with relatively little demyelination were observed in the spinal cord of C57BL/6 LT-{alpha}–/– (B) and C57BL/6 LT-ß–/– (C) mice. The lesions shown were some of the few in which a degree of demyelination was present accompanying the inflammation. Similar inflammatory foci were observed in the spinal cord of SJL mice (D), but the foci were associated with extensive demyelination. No lesions were seen in resistant C57BL/6 mouse (A).

 
Although the spinal cord of infected SJL mice had more extensive demyelination 42 days p.i. (Fig. 2D and Table 1) than that seen in C57BL/6 LT-{alpha}–/– mice (Fig. 2B and Table 1), the SJL mice failed to exhibit clinical signs at that time, while the C57BL/6 LT-{alpha}–/– mice became paralyzed. Therefore, the severity of clinical deficits was not correlated with the extent of demyelination, as has been previously observed in class I–/– and CD8–/– mice (6,28). The prominent clinical deficits of the LT-{alpha}–/– mice may be due to the presence of more severe inflammation in the gray matter of these mice compared to that seen in SJL mice (Table 1) or to the absence of functional CD8+ T cells which can contribute to the pathology (see later). These data suggest that the LT-regulated immune response may be important in clearing virus infection from the gray matter in addition to the white matter.

The effect of LT in DA-induced disease is via membrane LT-{alpha}1ß2 and not soluble LT-{alpha}3
Two forms of LT, a soluble LT-{alpha}3 and a membrane LT-{alpha}1ß2 respectively, bind TNF-R and LT-ß-R. To test the role of soluble LT-{alpha}3 in determining resistance to DA infection and DA-induced demyelination, C57BL/6 mice deficient in TNF-R1 or TNF-R2 were inoculated with virus. These mice failed to exhibit clinical signs, histopathology (Table 1) or evidence of virus antigen (data not shown) 42 days p.i., demonstrating that the soluble form of LT has no effect on resistance of C59BL/6 mice to TMEV disease. To test the role of membrane LT-{alpha}1ß2, we infected C57BL/6 mice deficient in LT-ß-R. Demyelination and inflammation with evidence of DA virus antigen were present in the spinal cord of these mice 42 days p.i.; although occasional lesions had evidence of inflammation associated with demyelination (Fig. 1F), most inflammatory foci were associated with relatively little demyelination compared to SJL mice, (Fig. 2C and Table 1). These findings suggest that membrane LT-{alpha}1ß2 signaling is critical in determining resistance to DA virus infection and also in mediating demyelination. In order to delineate whether the effect of membrane LT-{alpha}1ß2 is related to the absence of this protein during infection rather than the effects of LT-{alpha}1ß2 during immune system development, weanling mice infected with DA were treated with LT-ß-R–Ig fusion protein. No pathology (Fig. 1G and Table 1) or virus antigen (data not shown) was found 42 days p.i. in these treated mice, suggesting that the effect of membrane LT-{alpha}1ß2 on DA infection and demyelination is related to its action on early immune system development.

The absence of LT and TNF did not interfere with the production of DA virus-specific antibody following DA infection
LT plays an important role in antibody production (23). We therefore questioned whether the absence of LT signal or a deficiency of TNF impaired production of virus-specific antibody and led to disease in a normally resistant mouse strain. We performed an ELISA to test virus-specific Ig (IgG + IgM) in sera from infected mice 42 days p.i. No significant difference in virus-specific antibody levels was demonstrated among groups of wild-type C57BL/6, C57BL/6 LT-{alpha}–/– and C57BL/6 TNF–/– mice, and C57BL/6 mice treated with LT-ß-R–Ig (Fig. 3), suggesting that impaired antibody production was not responsible for the change in disease phenotype. However, the DA virus-specific antibody level was relatively low in infected C57BL/6 TNF-R1–/– mice compared to the other groups of infected mice, suggesting that the TNF-R1- mediated response may be important in optimizing the production of virus-specific antibody following DA virus infection. Since DA virus fails to persist and induce demyelination in C57BL/6 TNF-R1–/– mice, we conclude that other components of the immune system rather than virus-specific antibody can clear virus from the CNS (e.g. virus-specific CTL).



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Fig. 3. DA virus-specific Ig (IgG + IgM) as measured by ELISA in sera of mice 42 days p.i. There was no difference in virus-specific antibody production in the sera among wild-type C57BL/6, C57BL/6 LT-{alpha}–/– and C57BL/6 TNF–/– mice, and C57BL/6 mice treated with LT-ß-R–Ig; however, virus-specific antibody was significantly lower in C57BL/6 TNF-R1–/– mice.

 
The absence of LT and TNF did not interfere with NK cell activity in the CNS of mice following DA virus infection
The development of NK cells, a component of the host’s first line of defense against virus infection, has been shown to be impaired in LT-{alpha}–/– mice (29). In order to test whether NK cell activity was impaired in the CNS of mice following DA virus infection, we isolated CNS-IMNC for a NK cell assay from C57BL/6, C57BL/6 LT-{alpha}–/–, C57BL/6 TNF–/–, C57BL/6 TNF-R1–/–, C57BL/6 TNF-R2–/– and C57BL/6 LT-ß-R–/– mice and C57BL/6 mice treated with LT-ß-R–IgG 7 days p.i. The results showed that NK cell activity in the CNS was not impaired in the absence of LT and TNF following DA virus infection (data not shown), and suggested that NK cells do not play an important role in the susceptibility of LT-{alpha}–/– and LT-ß-R–/– mice to DA infection.

Virus-specific CTL activity in the CNS of C57BL/6 LT-{alpha}–/– and LT-ß-R–/– mice is impaired
Virus-specific CTL are a primary virus-specific effector system that is believed responsible for controlling DA virus infection in C57BL/6 and BALB/c mice (37). A failure to mount an anti-virus CTL response may be responsible for the susceptibility of SJL mice to DA infection (and to the late demyelinating disease). We questioned whether an absence of LT-{alpha}1ß2 could lead to a reduced number and/or impaired distribution of DC in lymphoid tissues with a subsequent impairment of antigen presentation, thus interrupting generation of virus-specific CTL in the CNS following DA virus infection. To test this hypothesis, CNS-IMNC were isolated 7 days p.i. from C57BL/6, C57BL/6 LT-{alpha}–/–, C57BL/6 LT-ß-R–/–, C57BL/6 TNF–/–, C57BL/6 TNF-R1–/– and C57BL/6 TNF-R2–/– mice, and from C57BL/6 mice treated with LT-ß-R–Ig. Virus-specific CTL were present in the CNS of all the mouse groups except for C57BL/6 LT-{alpha}–/– and C57BL/6 LT-ß-R–/– mice (Fig. 4). Virus-specific CTL activity was also present in isolated CNS-IMNC 7 days p.i. in C57BL/6 mice that had received an i.v. inoculation of LT-ß-R–Ig. These results indicated that the virus-specific CTL activity in the CNS of LT-{alpha}–/– and LT-ß-R–/– mice was severely impaired, and presumably led to a failure in virus clearance with resultant virus persistence and demyelination. Our findings suggest CD8+ T cell development and the subsequent generation of anti-virus CTL depend on membrane LT-{alpha}1ß2 which is perturbed in LT-{alpha}–/– and LT-ß-R–/– mice.



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Fig. 4. Virus-specific CTL activity in the CNS of DA virus-infected mice. CNS-IMNC were isolated from mice 7 days p.i. for a cytotoxicity assay. C57SV and C57SV/LP cells (which express DA virus leader, VP4, VP2 and VP3) were used as targets. Virus-specific cytotoxicity was severely impaired in C57BL/6 LT-{alpha}–/– and C57BL/6 LT-ß-R–/– mice, but not in TNF–/–, TNF-R1–/– and TNF-R2–/– mice or mice treated with LT-ß-R–Ig.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Interactions between LT and TNF with their receptors are important in the development of the immune system and in the activation of a wide variety of inflammatory responses. In this study we explored the role of LT and TNF in resistance/susceptibility to DA virus infection and virus-induced demyelination. We were especially interested in investigating the effect of LT and TNF on DA infection since the immune response is known to act in both clearing DA virus as well as mediating the virus-induced white matter disease (1,2).

LT is present in a soluble form as LT-{alpha}3 and a membrane form as LT-{alpha}1ß2. Soluble LT-{alpha}3 and the structurally related TNF-{alpha}3 induce similar activities by binding to TNF-R1 and TNF-R2. In contrast, membrane LT-{alpha}1ß2 binds to LT-ß-R (21). We used knockout mice to investigate the role of LT and TNF in a mouse strain that is normally resistant to DA virus infection. We found that LT-{alpha}–/– and LT-ß-R–/– mice, but not TNF–/– and TNF-R1–/– and R2–/– mice, developed persistent DA virus infection and DA virus-induced demyelination, indicating that membrane LT-{alpha}1ß2, but not soluble LT-{alpha}3 or TNF, plays a critical role in virus clearance in a mouse strain haplotype normally resistant to DA virus infection.

We hypothesized that the action of membrane LT-{alpha}1ß2 in mediating resistance to DA virus infection was related to its effects on CTL activity of CD8+ T cells since it is likely that MHC class I-restricted, virus-specific CD8+ T cells help determine resistance to TMEV infection (37,28). Our study demonstrated impaired virus-specific CTL activity in the CNS of C57BL/6 LT-{alpha}–/– and LT-ß-R–/– mice 7 days p.i, emphasizing the importance of membrane LT-{alpha}1ß2 in anti-virus CTL activity and resistance to DA infection. In contrast, virus-specific CTL activity was not impaired in the CNS of wild-type C57BL/6, TNF–/–, TNF-R1–/– and TNF-R2–/– mice, suggesting that signals from TNF-R1 and TNF-R2 (LT-{alpha}3 and TNF) as well as TNF are not involved in the development and function of CD8+ T cells.

Of interest, we found that resistant C57BL/6 mice that received LT-ß-R–Ig fusion protein (which blocks membrane LT-{alpha}1ß2 effect) during the acute virus infection behaved very differently from infected C57BL/6 LT-{alpha}–/– and LT-ß-R–/– mice, since weanling animals that received the fusion protein were able to clear virus, failed to demyelinate and generated virus-specific CTL activity in the CNS. These results suggested that the effect of membrane LT-{alpha}1ß2 on mediating resistance of weanling mice to DA virus infection and disease was not via the LT-ß-R during the acute virus infection, but was likely through membrane LT-{alpha}1ß2- LT-ß-R signaling on LT-ß-R-expressing cells during development of the immune system, and this effect was fixed by 3 weeks of age. LT-{alpha}–/–, LT-ß–/– and LT-ß-R–/– mice have profoundly defective lymph nodes and Peyer’s patches as well as an altered splenic microarchitecture because of an absence of membrane LT-{alpha}1ß2 during the second half of gestation (23,3036). The absence of lymph nodes and Peyer’s patches may fail to provide the appropriate co-stimulation environment for activation of CD8+ T cells. A disturbance in the development of the immune system is likely to perturb the generation of DA virus-specific CTL since T cells need to mature in the lymph nodes or spleen in order to carry out their effector function. LT is also important in the maturation of a DC subset that is critical for T cell function (37). The LT-mediated microenvironment is presumably developmentally fixed and therefore not reversed by treatment with LT-B-R–Ig fusion protein during life, as demonstrated in this report. LT-{alpha}–/– mice also have inadequate expression of the IL-12 receptor (19), which is a key factor in the differentiation of CD8+ T cells into potent effectors (20). Our data suggest that the absence of membrane LT-{alpha}1ß2-LT-ß-R signaling at an early stage of immune system development prevented the generation of anti-virus CTL following DA infection of LT-{alpha}–/– and LT-ß–/– C57BL/6 mice. The impairment in anti-virus CTL prevented virus clearance in this normally resistant mouse strain; DA virus persistence within the CNS set the stage for immune-mediated demyelination.

Several studies have emphasized the importance of an organized lymphoid system for the generation of effective levels of immunity to varied pathogenic agents (17,18,38), and highlighted the role of LT and TNF in the host defense against viruses. LT-{alpha}–/– mice have a markedly enhanced susceptibility to encephalitis following infection with herpes simplex virus (19). The infected mice had impaired cytotoxicity and expression of IFN-{gamma} by CD8+ T cells. Although the frequency and levels of perforin and Fas ligand of CD8+ T cells in LT-{alpha}–/– mice were the same as those in wild-type mice, the cells remained largely at a naive state, as judged by high expression of CD62 ligand (a marker for naive T cells) and failure to up-regulate activation or memory markers. In addition, few CD8+ T cells expressed the activation markers CD44high and CD25, and none expressed CD69. The scarcity of LT-{alpha}–/– cells expressing CD44high, the adhesion molecule considered to be involved in endothelial cell adhesion and extravascular transport (39), is likely to have limited homing of these cells and their access to extravascular sites of virus infection such as the CNS. Studies involving lymphocytic choriomeningitis virus (LCMV) have also reported a decreased virus-specific CD8+ T cell response in LT-{alpha}–/– and LT-ß–/– mice (17,18,38); however, these studies did not specifically identify the role of membrane LT-{alpha}1ß2 in the development of the immune system as underlying the functional abnormalities of CD8+ T cells. Transfer studies showed that impairment in the activation of LCMV-specific T cells in LT-{alpha}–/– mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LT-{alpha}–/– T cells (38). A recent report regarding human cytomegalovirus emphasized the importance of LT-ß-R and TNF-R1 signaling in induction of IFN-ß in virus-infected cells and the establishment of anti-viral activity (40).

Although infected LT-{alpha}–/– and LT-ß-R–/– mice had evidence of significant pathology 42 days p.i., the majority of inflammatory lesions were not associated with demyelination. This finding contrasts with lesions found in SJL mice, in which inflammation and demyelination are generally coupled. It may be that strain differences between these mice led to the differences in pathology. Another possibility is that LT plays a role in mediating demyelination. The relative decrease in demyelination in infected LT-{alpha}–/– and LT-ß-R–/– mice given the significant degree of inflammation may have been due to the impaired CD8+ T cell function we observed in these mice, since CD8+ T cells are believed to contribute to demyelination following DA infection (2,46,28). It is also possible that this discordance was related to disturbed CD4+ T cell function, since MHC class II-restricted CD4+ T cells, which depend on LT for their development and function, also contribute to DA-induced demyelination (2,3,6,8).

Conflicting results have been reported regarding the role of TNF in DA-induced demyelination (13,15,41,42). TNF is expressed in the CNS of mice following infection with TMEV (13,41) and a decrease in TNF expression or of TNF-expressing T cells has been correlated with decreased TMEV-induced demyelination (13,15,41). On the other hand, others have reported that administration of recombinant TNF-{alpha} inhibits DA virus-induced demyelination (42). Therefore, the importance of TNF in TMEV disease remains unclear, especially its effect on resistance/susceptibility to infection. We found that TNF–/–, TNF-R1–/– and TNF-R2–/– mice had no change in the resistant phenotype, indicating that TNF is not important in determining resistance to DA infection in C57Bl/6 mice. We suspect that DA virus-specific CTL, which were present in the CNS of TNF–/–, TNF-R1–/– and TNF-R2–/– mice, cleared virus in these mice as in the case of wild-type C57BL/6 mice. The reason for the preserved CD8+ T cell function in TNF–/–, TNF-R1–/– and TNF-R2–/– mice is presumably related to a normal number and distribution of splenic DC in these mice, in contrast to the situation with LT-{alpha}–/– and LT-ß-R–/– mice. TNF, but not LT, has been found to play a significant role in mediating the white matter disease in experimental allergic encephalomyelitis, an immune-mediated demyelinating disease that serves as a non-viral experimental model of multiple sclerosis (43). We could not specifically assess the role of TNF in the demyelination associated with DA infection, because DA virus failed to persist in TNF–/–, TNF-R1–/– and TNF-R2–/– mice, and virus persistence is necessary for demyelination to occur (2,6,10).

We questioned whether the action of LT or TNF on antibody production might influence resistance to DA virus infection since anti-virus antibody plays a role in DA virus clearance, and because previous studies showed that B cell function and distribution were impaired in LT-{alpha}–/– mice and LT-ß-R–Ig-treated mice (23,44). Published data suggest that: membrane LT-{alpha}1ß2 from lymphocytes may regulate non-lymphocytic cells that support formation of B cell follicles and T/B cell segregation in lymphoid tissue (23,44,45); LT may regulate the migration or distribution of bone marrow-derived cells into lymphoid tissue (44,46); mice deficient in LT-{alpha}, but not TNF, fail to generate both the primary and secondary IgG response to sheep red blood cells (SRBC); administration of LT-ß-R–Ig but not TNF-R1–Ig, prevents primary IgG production in response to SRBC challenge. We found, however, that levels of virus-specific antibody produced by LT-{alpha}–/– and LT-ß-R–Ig-treated mice were comparable to that seen in wild-type animals. There are a number of possible explanations as to why anti-virus antibody appeared normal in the DA-infected LT-{alpha}–/– and LT-ß-R–Ig-treated mice. Previous studies have measured systemic B cell function following peripheral SRBC inoculation, while our investigation involved a CNS immune response following intracerebral virus inoculation. It is also possible that the decreased SRBC antibody production reported in other studies is related to a decrease in a specific Ig subtype that is different from that produced following DA infection. We did find low levels of virus-specific antibody in sera of DA virus-infected TNF-R1–/– mice, suggesting that TNF-R1-mediated responses are important for production of virus-specific antibodies following intracerebral DA infection. However, these TNF-R1–/– mice did not develop persistent virus infection and demyelination, indicating that a severely impaired production of virus-specific antibody was not sufficient to disrupt the resistance of these mice to DA infection.

Our studies demonstrate the importance of membrane LT-{alpha}1ß2 in resistance to DA infection and suggest that its role in the development of the immune system allows for the later generation of anti-virus CD8+ T cells with subsequent virus clearance. Our studies also suggest that, in addition to fostering resistance to DA infection, membrane LT-{alpha}1ß2 is involved in mediating demyelinating pathology.


    Acknowledgements
 
We thank Vytas Bindokas for assistance with photography. This work was supported by grants from National Multiple Sclerosis Society (RG 3127-A-1 and RG 3068-A-1), and the National Institutes of Health (5 RO1 NS37958-04).


    Abbreviations
 
CNS—central nervous system

CTL—cytotoxic T lymphocyte

DA—Daniel’s strain of TMEV

DC—dendritic cell

IMNC—infiltrating mononuclear cell

LT—lymphotoxin

p.i.—post-infection

R—receptor

TMEV—Theiler’s murine encephalomyelitis virus

TNF—tumor necrosis factor

SRBC—sheep red blood cell


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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