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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: cytokine, cytotoxic T lymphocyte, demyelination, lymphotoxin, Theilers virus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LT, which is primarily expressed in activated T, B and NK cells (21,22), is present in two forms, soluble LT-3 homotrimers and membrane-associated LT-
1ß2 heterotrimers. Soluble LT-
3 is structurally related to TNF-
3 and binds either of two TNF receptors, TNF-R1 and TNF-R2. Soluble LT-
3 and TNF-
3 induce similar effects by activating a wide variety of inflammatory and immune responses. In contrast, membrane LT-
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-/ (which are deficient in both soluble LT-
3 and membrane LT-
1ß2), LT-ß/ (which are deficient in membrane-associated LT-
1ß2) and LT-ß-R/ mice have profoundly defective development of the peripheral lymphoid organs (21). LT-
/ mice and mice in which membrane LT-
1ß2 has been blocked by an LT-ß-RIg 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-1ß2, but not soluble LT-
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-
1ß2 was not only important in mediating resistance to TMEV infection, but also in fostering the demyelinating pathology.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Female C57BL/6 mice (46 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME); 4- to 6-week-old female C57BL/6 LT-/ mice deficient in both soluble LT-
3 and membrane LT-
1ß2, C57BL/6 LT-ß-R/ mice deficient in the receptor for membrane LT-
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-RIg fusion protein, which binds LT-1ß2 and blocks the effect of membrane LT-
1ß2, was prepared as previously described (25). Selected mice received i.p. inoculation(s) of 100 µg LT-ß-RIg 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 Students 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 Students t-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (both soluble LT-
3 and membrane LT-
1ß2) and TNF. Following DA infection, three of seven C57BL/6 LT-
/ 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-
, but not TNF. The data suggest that LT-
, 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-/ 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-
/ mice, but not TNF/ mice or wild-type C57BL/6 (data not shown). Therefore, LT-
, 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-/ 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-
/ 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-
/ compared to SJL mice, the lesions in C57BL/6 LT-
/ 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.
|
|
|
The effect of LT in DA-induced disease is via membrane LT-1ß2 and not soluble LT-
3
Two forms of LT, a soluble LT-3 and a membrane LT-
1ß2 respectively, bind TNF-R and LT-ß-R. To test the role of soluble LT-
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-
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-
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-
1ß2 is related to the absence of this protein during infection rather than the effects of LT-
1ß2 during immune system development, weanling mice infected with DA were treated with LT-ß-RIg 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-
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-/ and C57BL/6 TNF/ mice, and C57BL/6 mice treated with LT-ß-RIg (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).
|
Virus-specific CTL activity in the CNS of C57BL/6 LT-/ 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-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-
/, 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-RIg. Virus-specific CTL were present in the CNS of all the mouse groups except for C57BL/6 LT-
/ 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-RIg. These results indicated that the virus-specific CTL activity in the CNS of LT-
/ 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-
1ß2 which is perturbed in LT-
/ and LT-ß-R/ mice.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LT is present in a soluble form as LT-3 and a membrane form as LT-
1ß2. Soluble LT-
3 and the structurally related TNF-
3 induce similar activities by binding to TNF-R1 and TNF-R2. In contrast, membrane LT-
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-
/ 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-
1ß2, but not soluble LT-
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-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-
/ and LT-ß-R/ mice 7 days p.i, emphasizing the importance of membrane LT-
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-
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-RIg fusion protein (which blocks membrane LT-1ß2 effect) during the acute virus infection behaved very differently from infected C57BL/6 LT-
/ 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-
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-
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-
/, LT-ß/ and LT-ß-R/ mice have profoundly defective lymph nodes and Peyers patches as well as an altered splenic microarchitecture because of an absence of membrane LT-
1ß2 during the second half of gestation (23,3036). The absence of lymph nodes and Peyers 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-RIg fusion protein during life, as demonstrated in this report. LT-
/ 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-
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-
/ 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-/ 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-
by CD8+ T cells. Although the frequency and levels of perforin and Fas ligand of CD8+ T cells in LT-
/ 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-
/ 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-
/ and LT-ß/ mice (17,18,38); however, these studies did not specifically identify the role of membrane LT-
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-
/ mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LT-
/ 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-/ 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-
/ 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- 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-
/ 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-/ mice and LT-ß-RIg-treated mice (23,44). Published data suggest that: membrane LT-
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-
, but not TNF, fail to generate both the primary and secondary IgG response to sheep red blood cells (SRBC); administration of LT-ß-RIg but not TNF-R1Ig, prevents primary IgG production in response to SRBC challenge. We found, however, that levels of virus-specific antibody produced by LT-
/ and LT-ß-RIg-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-
/ and LT-ß-RIg-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-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-
1ß2 is involved in mediating demyelinating pathology.
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
CTLcytotoxic T lymphocyte
DADaniels strain of TMEV
DCdendritic cell
IMNCinfiltrating mononuclear cell
LTlymphotoxin
p.i.post-infection
Rreceptor
TMEVTheilers murine encephalomyelitis virus
TNFtumor necrosis factor
SRBCsheep red blood cell
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|