JOURNAL OF VIROLOGY, Mar. 2000, p. 2447–2450 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
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Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System BEATRIZ PARRA,1 MARK T. LIN,2 STEPHEN A. STOHLMAN,1,3* CORNELIA C. BERGMANN,1,3 ROSCOE ATKINSON,2,3 AND DAVID R. HINTON2,3 Departments of Molecular Microbiology and Immunology,1 Pathology,2 and Neurology,3 Keck School of Medicine, University of Southern California, Los Angeles, California 90033 Received 20 September 1999/Accepted 30 November 1999
The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication. CD8⫹ T cells are crucial for viral clearance during acute central nervous system (CNS) infection induced by the neurotropic JHM strain of mouse hepatitis virus (JHMV) (20). In general, CD8⫹ T cells exert antiviral effector functions via two basic mechanisms: a nonlytic pathway involving cytokines (2, 18) and two mechanisms based on cytotoxicity (10, 12, 13). Major histocompatibility complex (MHC) class I-dependent lysis of infected cells via release of perforin-containing granules is the main CD8⫹-T-cell cytotoxic pathway (10, 11). However, cell-cell interactions between Fas, expressed on infected cells, and Fas ligand (FasL), expressed on activated T cells, can also lead to cytolysis (17). JHMV clearance from the CNS of gamma interferon (IFN-␥)- or perforin-deficient mice demonstrated that both lytic and nonlytic mechanisms contribute to controlling virus replication in a cell-type-dependent manner (14, 16). Failure of IFN-␥-deficient mice to clear virus from oligodendrocytes suggested a critical role of this cytokine in clearance from this cell type (16). On the other hand, analysis of perforin-deficient mice demonstrated that this cytolytic effector mechanism is important in controlling replication in microglia and astrocytes (14). Although delayed, infectious virus is completely eliminated from the CNS of perforin-deficient mice. This suggested that perforin-mediated cytolysis is a dispensable mechanism for JHMV clearance (14). A reduction in the majority of infectious virus in the absence of IFN-␥ and delayed clearance in the absence of perforin confirmed a partial but not exclusive contribution of perforin-dependent cytotoxicity and suggested an additional antiviral effector mechanism(s). Fas-FasL interactions regulate a major pathway of apoptosis, which may play an important role in both mediating antiviral effects and the pathogenesis of autoimmune CNS diseases. Although Fas is not normally expressed on CNS cells, its expression is upregulated during inflammation (5–7, 19). To determine if Fas-FasL interactions contribute to the pathogenesis of JHMV infection in the CNS, Fas-deficient B6.MRLFaslpr (lpr) mice (Jackson Laboratories, Bar Harbor, Maine) and congenic wild-type (wt) C57BL/6J mice (Jackson Laboratories) were infected at 6 to 7 weeks of age with 200 PFU of the neutralizing monoclonal antibody-derived antigenic JHMV variant, designated 2.2v-1 (8, 9, 14, 16). The levels of virus
replication and clinical disease were compared in both groups for 21 days postinfection (p.i.). Mice in both groups exhibited signs of acute encephalitis and paralysis followed by slowly progressive, but almost complete, recovery at day 21 p.i. lpr mice exhibited neither differences in disease severity nor mortality following infection in comparison to wt mice. Analysis of the kinetics of JHMV clearance from the CNS, determined by plaque assay on DBT cells (14), showed a high level of virus replication 3 days p.i. in both lpr and wt mice. Viral titers subsequently declined in both groups until day 12 p.i. (Fig. 1), after which infectious virus could no longer be recovered. Therefore, similar kinetics of virus replication and clearance were found in lpr mice and wt mice (Fig. 1). In contrast to the absence of perforin-dependent cytolysis (14) and IFN-␥ (16), these data suggest that Fas-FasL interactions do not contribute significantly to JHMV clearance when perforin- or IFN-␥-mediated pathways are functional. However, it is a possibility that Fas-mediated cytolysis may counteract the loss of perforin-mediated cytolytic activity by the infiltrating virus-specific CD8⫹ T cells at late stages of infection (4). To determine whether perforin-mediated cytolysis or IFN-␥ secretion compensated for the absence of Fas-FasL interac-
* Corresponding author. Mailing address: University of Southern California, Keck School of Medicine, Department of Neurology, 1333 San Pablo St., MCH 142, Los Angeles, CA 90033. Phone: (323) 4421063. Fax: (323) 225-2369. E-mail:
[email protected].
FIG. 1. Comparison of the kinetics of JHMV replication in the brains of B6.MRL-Faslpr (lpr) and C57BL/6 (wt) mice. Mice were infected with 200 PFU of JHMV. Virus titers were determined by plaque assay, and each time point p.i. represents the mean titer for a group of at least three mice.
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FIG. 2. Fas-deficient B6.MRL-Fas (lpr) (B, D, and F) and wild-type (wt) C57BL/6J (A, C, and E) mice were infected with 200 PFU of JHMV and sacrificed at days 7, 12, and 21 p.i. CNS tissues were fixed in 75% ethanol–25% glacial acetic acid and embedded in paraffin. (A and B) At day 7, prominent perivascular inflammatory infiltrates (arrowheads) were found. (C and D) At day 21 p.i., only scattered foci of inflammatory cells (arrowheads) were found; perivascular infiltrates were no longer present. (E and F) Similar numbers of apoptotic cells (arrowheads), shown here in the spinal cord at day 21 p.i., were found in the brains and spinal cords of both wt and lpr mice. (A to D) Hematoxylin and eosin stain; (E and F) terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling stain. Bars, 200 m.
tions, the magnitudes and effector functions of virus-specific CD8⫹ T cells were compared in the groups of infected mice. Cytotoxic T-lymphocyte (CTL) activity in splenocytes was measured by a 51Cr release assay after 6 days of in vitro stimulation (3, 14). The levels of specific IFN-␥-secreting CD8⫹ T cells in CNS mononuclear cell infiltrates, cervical lymph nodes, and splenic cells were compared by the ELISPOT assay (4). Finally, virus-specific CD8⫹ T cells within the brain mononuclear cell infiltrates were quantitated by fluorescence-activated cell
sorter analysis by using a tetrameric MHC-Db-510 peptide complex as previously described (4). However, no differences were found in either the frequency of IFN-␥-secreting virusspecific CD8⫹ T cells or in the CTL activity specific for the S510 epitope between lpr mice and wt mice (data not shown). In addition, similar proportions of virus-specific CD8⫹ T cells infiltrated the CNS of both Fas-deficient and infected wt mice (data not shown). Fas-FasL interactions play two potential roles in the patho-
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genesis of CNS diseases. First, activation-induced T-cell apoptosis is mediated via Fas (1, 5), suggesting a role in regulating mononuclear cell infiltration during resolution of acute infection. Second, Fas-FasL interactions are associated with autoimmune CNS demyelination (6, 7, 22), possibly via oligodendrocyte apoptosis (7). To determine whether Fas-mediated cytotoxicity contributes to JHMV-induced inflammation, mononuclear cell infiltrates, levels of demyelination, and the frequencies of apoptotic cells were compared in infected lpr mice and wt mice. Analysis of the brain and spinal cord revealed no differences in the extent or distribution of inflammatory cells at any time point examined. Mononuclear cell infiltrates were prominent in the brain at 7 days p.i. (Fig. 2A and B) and in the brain and spinal cord at days 12, 14, and 16 p.i. No perivascular mononuclear cell infiltrates remained at day 21 p.i. Although diffuse foci of mononuclear cells were still present in the parenchyma at 21 days p.i., there was no difference between the two groups (Fig. 2C and D). Although numerous apoptotic infiltrating mononuclear cells were present in the CNS of Fas-deficient and wt mice at days 12 and 16, no differences in the amount or distribution of apoptotic cells were observed. The number of apoptotic cells declined with time, and even at day 21 p.i., no differences were found (Fig. 2E and F). Furthermore, no differences were found in either the distribution or frequency of CD4⫹ or CD8⫹ T cells when wt and Fas-deficient mice were compared by immunohistochemistry (data not shown), consistent with the analysis of both total and antigen-specific CD8⫹ T cells derived from the CNS mononuclear cell population. These data suggest that downregulation of immunopathology during acute JHMV infection of the CNS is not exerted primarily through Fas-mediated apoptosis. This is consistent with the Fas-independent apoptosis of inflammatory cells observed during LCMV infection (15). Extensive but equal levels of demyelination were also present in both groups at days 14, 16, and 21 p.i. (data not shown), suggesting that Fas-mediated cytolysis does not contribute to JHMV-induced demyelination. Consistent with equivalent levels of demyelination, the identical cellular localization of viral antigens in both groups suggested that, in contrast to the results with mice deficient in either IFN-␥ (16) or perforin (14), Fas-mediated cytolysis does not regulate JHMV replication in a cell-type-specific manner. The indistinguishable responses of Fas-deficient and wt mice to JHMV infection support the concept that the Fas-FasL cytolytic mechanism does not contribute to the resolution of virus-induced inflammation. Fas-dependent cytolysis thus either is a dispensable anti-JHMV immune effector mechanism or predominantly contributes to virus clearance from the CNS during the loss of perforin-mediated cytolysis (4). To examine the role of Fas-dependent cytolysis in the absence of perforin, JHMV replication was analyzed in chimeric mice, prepared as described by Topham et al. (21). Briefly, lpr and wt mice were lethally irradiated (950 rads) and immediately reconstituted with 7 ⫻ 107 naive splenocytes from either perforin-deficient (P⫺/⫺) (Jackson Laboratories) or syngeneic wt (P⫹/⫹) mice (termed reconstituted mice). Four days after reconstitution, mice were infected with JHMV. All mice developed clinical disease (encephalitis and limb paralysis) following JHMV infection. However, in contrast to wt mice, irradiated chimeric mice exhibited a higher level of mortality and did not recover from disease. Experiments were terminated at day 13 p.i. when the mortality in the control chimeric wt group (Fas⫹/⫹/P⫹/⫹) approached 80%. Virus titers in reconstituted wt mice (Fas⫹/⫹/P⫹/⫹) were higher at 6 days p.i. (Fig. 3) than those in untreated wt mice (Fig. 1), demonstrating that replication is enhanced at early
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FIG. 3. JHMV replication in the CNS of chimeric mice. Virus titers in brains of reconstituted wt mice (Fas⫹/⫹/P⫹/⫹) (A), Fas-deficient mice (Fas⫺/⫺/P⫹/⫹) (B), Fas-competent mice (Fas⫹/⫹/P⫺/⫺) (C), and Fas- and perforin-deficient mice (Fas⫺/⫺/P⫺/⫺) (D) following infection with 200 PFU of JHMV are shown. Virus titers below the detection level (⬍26 PFU/g of brain tissue) are expressed as 0 values.
time points in the irradiated-reconstituted wt recipients in comparison to that in untreated wt mice. Virus was eliminated from the CNS of 75% of the reconstituted wt mice by day 11 p.i. and from 80% of these mice by day 13 p.i., the last time point examined, consistent with the kinetics of viral clearance in untreated wt mice (Fig. 1). By contrast, the majority of chimeric mice with only either the Fas (Fas⫹/⫹/P⫺/⫺) or the perforin (Fas⫺/⫺/P⫹/⫹) pathway intact still harbored significant virus titers at day 11 p.i. (Fig. 3). The disparity between delayed viral clearance in Fas⫺/⫺/P⫹/⫹ mice and that in lpr mice may be attributed to the higher virus load at day 5 p.i. in all reconstituted mice. However, virus was essentially eliminated from the CNS of both the Fas⫹/⫹/P⫺/⫺ and Fas⫺/⫺/P⫹/⫹ reconstituted groups by day 13 p.i., indicating delayed viral clearance in comparison to that of the wt reconstituted group. These data suggest that neither perforin- nor Fas-dependent cytolysis alone is an absolute requirement for clearance of JHMV from CNS, similar to the Fas-dependent clearance of influenza virus (21). In contrast to mice deficient in a single pathway, chimeric mice lacking both pathways were unable to control infectious virus (Fig. 3D). These results suggest that in the absence of one cytolytic pathway, the remaining lytic mechanism is sufficient to control JHMV replication in CNS. Histological analysis of infected chimeric mice showed maximal mononuclear cell infiltration at day 9 p.i., with a slight decrease by day 13 in all groups despite the absence of either cytolytic mechanism. Similar levels of demyelination were present in all four chimeric groups. Therefore, the similar extents of both inflammation and demyelination in chimeric mice revealed no correlation between the absence of Fasdependent cytolysis and the accumulation of inflammatory cells or decreased CNS demyelination (data not shown). These data are consistent with the pathogenesis of JHMV in lpr mice (Fig. 2) and suggest that Fas-dependent cytotoxicity is not required for either inhibition of the extent of mononuclear cell
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infiltration or demyelination during JHMV-induced acute encephalomyelitis. Moreover, normal viral clearance in lpr mice (Fig. 1) and eventual viral clearance in chimeric Fas⫺/⫺/P⫹/⫹ mice, but not in chimeric Fas⫺/⫺/P⫺/⫺ mice (Fig. 3), confirm that in the presence of an intact perforin pathway, Fas-dependent cytolysis is redundant. However, the absence of perforin clearly revealed a functional antiviral role of Fas-dependent cytotoxicity in the CNS. The data suggest that Fas-FasL interactions are an alternative cytotoxic pathway that may operate predominantly in the absence of perforin-dependent cytolysis, which is downregulated during the clearance of infectious virus from the CNS (4). We thank Wen-Qiang and Chung Kang Ho for excellent technical assistance. This work was supported by grant NS 18146 from the National Institutes of Health. Beatriz Parra was supported by a training grant from Colciencias and Universidad del Valle (Cali, Colombia). REFERENCES 1. Alderson, M. R., T. W. Tough, T. Davis-Smith, S. Braddy, B. Falk, K. A. Schooley, R. G. Goodwin, C. A. Smith, F. Ramsdell, and D. H. Lynch. 1995. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med. 181:71–77. 2. Biron, C. A. 1994. Cytokines in the generation of immune responses to, and resolution of, virus infection. Curr. Opin. Immunol. 6:530–538. 3. Bergmann, C. C., Q. Yao, M. Lin, and S. A. Stohlman. 1996. The JHM strain of mouse hepatitis virus induces a spike protein-specific Db-restricted cytotoxic T cell response. J. Gen. Virol. 77:315–325. 4. Bergmann, C. C., J. D. Altman, D. Hinton, and S. A. Stohlman. 1999. Inverted immunodominance and impaired cytolytic function of CD8⫹ T cells during viral persistence in the central nervous system. J. Immunol. 163:3379– 3387. 5. Bonetti, B., J. Pohl, Y.-L. Gao, and C. S. Raine. 1997. Cell death during autoimmune demyelination: effector but not target cells are eliminated by apoptosis. J. Immunol. 159:5733–5741. 6. Dowling, P., G. Shang, S. Raval, J. Menonna, S. Cook, and W. Husar. 1996. Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain. J. Exp. Med. 184:1513–1518. 7. D’Souza, S. D., B. Bonnetti, V. Balasingam, N. R. Cashman, P. A. Barker, A. B. Troutt, C. S. Raine, and J. P. Antel. 1996. Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184:2361–2370. 8. Fleming, J. O., M. D. Trousdale, F. A. K. el-Zaatari, S. A. Stohlman, and
9.
10. 11.
12. 13.
14. 15. 16. 17. 18. 19.
20.
21. 22.
L. P. Weiner. 1986. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol. 58:869–875. Fleming, J. O., S. A. Stohlman, R. C. Harmon, M. M. C. Lai, J. A. Frelinger, and L. P. Weiner. 1983. Antigenic relationship of murine coronavirus: analysis using monoclonal antibodies to JHM (MHV-4) virus. Virology 131:296– 307. Henkart, P. A. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1:343–346. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31–37. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, et al. 1994. Fas and perforin pathways as major mechanisms of T-cell-mediated cytotoxicity. Science 265:528–530. Kagi, D., P. Seiler, P. Pavlovic, B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengatner. 1995. The roles of perforin and fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses. Eur. J. Immunol. 25:3256–3262. Lin, M. T., S. A. Stohlman, and D. R. Hinton. 1997. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis. J. Virol. 71:383–391. Lohman, B. L., E. S. Razvi, and R. M. Welsh. 1996. T-lymphocyte downregulation after acute viral infection is not dependent on CD95(Fas) receptor-ligand interactions. J. Virol. 70:8199–8203. Parra, B., D. R. Hinton, N. W. Marten, C. C. Bergmann, M. T. Lin, C. S. Yang, and S. A. Stohlman. 1999. IFN-␥ is required for viral clearance from central nervous system oligodendroglia. J. Immunol. 162:1641–1647. Rouvier, R., M.-F. Luciani, and P. Golstein. 1993. Fas involvement in Ca2⫹independent T-cell mediated cytotoxicity. J. Exp. Med. 177:195–200. Ruby, J., and I. Ramshaw. 1991. The antiviral activity of immune CD8⫹ T cells is dependent on interferon-␥. Lymphokine Cytokine Res. 10:353–358. Sabelko, K. A., K. A. Kelly, M. H. Nahm, A. H. Cross, and J. H. Russell. 1997. Fas and Fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system. J. Immunol. 159:3096–3099. Stohlman, S. A., C. C. Bergmann, R. C. van der Veen, and D. R. Hinton. 1995. Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from oligodendroglia. J. Virol. 69:684–694. Topham, D. J., R. A. Tripp, and P. C. Doherty. 1997. CD8⫹ T cells clear influenza virus by perforin or Fas-dependent processes. J. Immunol. 159: 5197–5200. Waldner, H., R. A. Sobel, E. Howard, and V. K. Kuchroo. 1997. Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis. J. Immunol. 159:3100–3103.
