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The Journal of Immunology, 1999, 162: 6543-6551.
Copyright © 1999 by The American Association of Immunologists

Potential Role of CD4+ T Cell-Mediated Apoptosis of Activated Astrocytes in Theiler’s Virus-Induced Demyelination1

JoAnn P. Palma, Robert L. Yauch2, Sharon Lang and Byung S. Kim3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracerebral inoculation of Theiler’s murine encephalomyelitis virus (TMEV) into susceptible mouse strains results in a chronic, immune-mediated demyelinating disease similar to human multiple sclerosis. Here, we examined the role of astrocytes as an APC population in TMEV-induced demyelination and assessed the potential consequences of T cell activation following Ag presentation. IFN-{gamma}-pretreated astrocytes were able to process and present all the predominant T cell epitopes of TMEV to virus-specific T cell hybridomas, clones, as well as bulk T cells. Despite low levels of proliferation of T cells due to prostaglandins produced by astrocytes, such Ag presentation by activated astrocytes induced the production of IFN-{gamma}, a representative proinflammatory cytokine, in TMEV-specific Th cell clones derived from the CNS of virus-infected mice. Furthermore, these Th cell clones mediate lysis of the astrocytes in vitro in a Fas-dependent mechanism. TUNEL staining of CNS tissue demonstrates the presence of apoptotic GFAP+ cells in the white matter of TMEV-infected mice. These results strongly suggest that astrocytes could play an important role in the pathogenesis of TMEV-induced demyelination by activating T cells, subsequently leading to T cell-mediated apoptosis of astrocytes and thereby compromising the blood-brain barrier.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the etiology of multiple sclerosis (MS)4 remains unknown, many epidemiological studies strongly suggest the potential involvement of an infectious agent(s) (1, 2, 3). Several animal models have been extensively used to investigate the nature of the pathogenic mechanisms of demyelinating diseases induced by viruses as the causative agent. In particular, the intracerebral inoculation of Theiler’s murine encephalomyelitis virus (TMEV) into susceptible strains of mice results in a chronic demyelinating disease that shares many of the features of human MS (4). As with human MS, the pathogenesis of TMEV-induced demyelinating disease (TMEV-IDD) seems to be immune-mediated, based on immunologic, histopathologic, and genetic evidence (5, 6, 7, 8, 9). Due to such close similarities, TMEV-IDD has served as a relevant infectious animal model for studying MS.

TMEV-specific T lymphocytes play a critical role in the immune-mediated tissue damage following viral infection. Demyelinating lesions are characterized by inflammatory cell infiltrates consisting of T cells as well as macrophages (10, 11), and the course of disease correlates with the development of a virus-specific Th1 response (12). In addition, the treatment with Abs to either the class II or CD4 molecules can significantly suppress demyelination induced by TMEV (6, 13). Class I-restricted T cells appear to be involved in the protection, rather than the pathogenesis of demyelinating disease (14, 15). Collectively, these results strongly support the involvement of virus-specific, class II-restricted CD4+ T cells in the pathogenesis of TMEV-IDD.

The local presentation of viral epitopes to virus-specific CD4+ T lymphocytes infiltrating the CNS may be a critical step in initiating and propagating the immunopathologic tissue damage following viral infection. MHC class II molecules are not normally expressed on CNS cell populations; however, they can be expressed on neuroglia cells during inflammatory conditions (16, 17), including TMEV-IDD and experimental autoimmune encephalomyelitis (EAE) (18, 19, 20). In particular, astrocytes can express MHC class II Ags following exposure to the proinflammatory cytokine IFN-{gamma} (21) or virus particles (22). Interestingly, the inducibility of class II expression on astrocytes by IFN-{gamma} appears to correlate with the mouse strain susceptibility to EAE (23) and TMEV-IDD (24), suggesting that the ability of astrocytes to present Ags to T cells may play an important role in the pathogenesis of demyelination. In vitro, IFN-{gamma}-treated astrocytes can process and present various protein Ags to T lymphocytes, including autoantigens (25) and conventional protein Ags (24, 26, 27). In addition, activated astrocytes can produce a variety of immunoregulatory cytokines (28, 29), as well as adhesion and costimulatory molecules (20, 27, 30), further suggesting an important role for these cells in immunopathological reactions of the CNS.

In this study, we analyzed the role of astrocytes as an APC population in the CNS during TMEV-IDD. We demonstrate here that IFN-{gamma}-treated astrocytes could effectively present all the major TMEV epitopes to virus-specific T cell hybridomas/clones and bulk T cells from virus-infected mice. Although prostaglandins released by the activated astrocytes could inhibit the proliferation of virus-specific T cell clones, such astrocytes were able to induce the production of IFN-{gamma} by these T cells. Interestingly, astrocytes involved in the T cell activation underwent Fas-mediated apoptosis by the virus-specific CD4+ T cells. Furthermore, significant levels of glial fibrillary acidic protein-expressing (GFAP+) apoptotic cells were seen in the CNS of infected mice. Since astrocytes are known to play an integral role in maintaining the blood-brain barrier (31, 32), such a destruction of astrocytes by activated virus-specific T cells may contribute to immune-mediated pathogenesis of TMEV-induced demyelination.


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

Young (4- to 6-wk-old) and pregnant (12 to 16 days gestation) female SJL/J mice were purchased from Charles River Breeding Laboratory (Wilmington, MA). Mice were subsequently housed in the animal care facility at Northwestern University (Chicago, IL).

Viruses and peptides

TMEV strain BeAn 8386 was propagated on BHK-21 cells grown in DMEM supplemented with 7% donor calf serum and purified by isopyknic centrifugation on Cs2SO4 gradients, as previously described (33). Purified TMEV was inactivated by exposure to a UV light source for 60 min at 4°C. The complete inactivation of TMEV by irradiation was verified by lack of viral plaque formation on BHK-21 cells. Synthetic peptides were prepared using the RaMPS peptide synthesis sytem (DuPont, Wilmington, DE). The amino acid sequences of peptides used in this study are: VP1233–250, SASVRIRYKKMKVFCPRP; VP274–86, QEAFSHIRIPLPH; VP324–37, PIYGKTISTPSDYM; and HEL34–45, FESNFNTQATNR.

Isolation of astrocytes

Astrocytes were isolated from the brains of 1- to 3-day-old SJL/J mice, as previously described (34). Briefly, brains were removed aseptically from newborn mice and freed from meninges, and single-cell suspensions were prepared by teasing tissue through sterile wire screens. Brain cells were washed several times before seeding in 75-cm2 flasks in DMEM supplemented with 10% FCS (Atlanta Biologicals, Norcross, GA) and gentamicin (Life Technologies, Grand Island, NY). Cells reached confluency ~8–14 days after seeding. At this time, flasks were shaken on a rotary platform at 250 rpm for 24 h to remove oligodendrocytes and microglia. Adherent astrocytes were trypsinized and replated. This procedure resulted in >90% astrocytes, as determined by staining with an Ab against GFAP (Sigma, St. Louis, MO). For use in proliferation or cytotoxicity assays, astrocytes were cultured for 24–48 h with 100 U/ml recombinant murine IFN-{gamma} (Genentech, San Francisco, CA). Astrocytes were subsequently washed three times before use in assays.

T cell hybridomas

Spleen cells (6 x 106 cells/ml) from virus-infected mice were cultured for 4 days in the presence of UV-inactivated TMEV (10–25 µg/ml) and subsequently fused using polyethylene glycol (m.w. 1450) to the TCR {alpha}ß- variant, BW5147 (35). For generation of T cell hybridomas from virus-immunized mice, a single-cell suspension of the lymph nodes from SJL/J mice immunized two times with 50 µg of purified UV-inactivated TMEV was prepared and further stimulated in vitro for 4 days with UV-inactivated TMEV (5 µg/ml). Resulting hybridomas were selected in hypoxanthine-aminopterin-thymidine medium, as previously described (36). All virus-specific hybridomas were subcloned by limiting dilution to assure clonality.

IL-2 production by T cell hybridomas

Hybridomas were tested for Ag specificity based on the production of IL-2 after stimulation with Ag in the presence of syngeneic APC. The production of IL-2 by hybridomas was determined by the ability of culture supernatants to support the growth of an IL-2-dependent cell line, CTLL-2. Briefly, 100 µl of supernatant from Ag-stimulated cultures were added to wells of microtiter plates containing 5 x 103 CTLL-2 cells in 100 µl of DMEM supplemented with 5% FCS and 5 x 10-5 M 2-ME. Cultures were incubated for 24 h and subsequently pulsed with 1 µCi/well of [3H]TdR (ICN Biochemicals, Costa Mesa, CA). Approximately 16 to 18 h later, cells were harvested, and [3H]TdR incorporation was determined in a liquid scintillation counter (Packard, Meriden, CT). Results are expressed as the mean cpm of triplicate cultures ± the SEM.

Establishment of T cell clones

Ag-specific T cell clones were established from the spinal cords of TMEV-infected SJL/J mice, as previously described (10). Briefly, mice were perfused with PBS to remove contaminating PBL, and single-cell suspensions of the spinal cords were isolated on a 100/50% discontinuous Histopaque gradient. Cells were then cultured on 96-well round-bottom microtiter plates, with UV-inactivated virus or peptides, in the presence of irradiated syngeneic splenocytes and 10 U/ml of IL-2 (Genzyme, Cambridge, MA). After TMEV-specific T cell lines were established, cells were subsequently cloned by limiting dilution. T cell clones were maintained by biweekly stimulation with UV-inactivated virus or peptides in the presence of syngeneic, splenocyte APCs and 5 U/ml IL-2.

T cell proliferation assay

Proliferation assays were conducted using either 5 x 105 irradiated, syngeneic splenocytes or various numbers of IFN-{gamma}-treated or untreated astrocytes as APCs. Briefly, 3 x 104 Histopaque-purified T cell clones were cultured for 72 h with different concentrations of UV-inactivated virus or peptides in the presence of various APCs. Cultures were pulsed with [3H]TdR, incubated for 16–18 h, harvested, and analyzed for [3H]TdR uptake, as described earlier.

Assay for IFN-{gamma} production

IFN-{gamma} concentrations were determined by capture ELISA. ELISA plates (Nunc, Naperville, IL) were coated overnight with 2 µg/ml of anti-IFN-{gamma} (ATCC, clone R4-6A2) and then blocked with PBS containing 1% nonfat dry milk. Plates were washed three times between steps with PBS containing 0.05% Tween 20. Culture supernatants were added to the plates and incubated overnight at 4°C. Concentrations of IFN-{gamma} in the culture supernatants were compared with that of recombinant murine IFN-{gamma} standard (Genentech). A polyclonal rabbit anti-mouse IFN-{gamma} Ab (Biosource, Camarillo, CA) was used as the detecting Ab. After consecutive incubations with biotinylated goat anti-rabbit IgG and streptavidin-HRP (Zymed, San Francisco, CA), plates were developed using the enzyme substrate 3,3',5,5'-tetramethylbenzidine (Dako, Carpinteria, CA) and read at 450 nm.

Cytolysis of astrocytes

Astrocyte lysis by CD4+ T cell clones or spinal cord cell suspension was measured by a standard 51Cr-release assay, as described previously (37). Astrocytes were grown to confluency (~1 x 104 cells/well) on 96-well round-bottom microtiter plates. Three days before the assay, 100 U/ml of IFN-{gamma} were added to the cultures. Cells were labeled overnight with 3 µCi/well of 51Cr in a total volume of 200 µl. On the day of the assay, adherent astrocyte monolayers were washed several times in BSS and subsequently pulsed with 1 µM peptide Ags. After 30 min, various numbers (3 x 105, 1 x 105, 5 x 104, and 1 x 104) of T cell clones were added to triplicate wells, corresponding to approximate E:T ratios of 30:1, 10:1, 5:1, and 1:1, respectively. For inhibition studies, anti-TNF-{alpha} Ab (1 mg/ml), Fas-Fc serum from transgenic mice (38), or control serum (2%) was added to the astrocyte cultures and assayed for 51Cr release. Lysis of IFN-{gamma}-activated astrocytes by effector cells in spinal cord cell suspensions from virus-infected animals was similarly assessed. The effector cells from spinal cords were prepared from virus-infected and uninfected animals, following perfusion and separation as described above. Cells at the interface of Histopaque gradient were counted and then added to the 51Cr-labeled target astrocytes with different peptide Ags. Controls included astrocytes cultured alone (spontaneous release) and astrocytes cultured with 1% Triton X-100 lysis solution (maximum release). After addition of effector cells, plates were centrifuged at 250 x g for 4 min and subsequently cultured for 5 h before analyzing 51Cr release. Sixty microliters of cell-free supernatant from each well were counted in the Top Count microplate scintillation counter (Packard). The specific lysis was determined based on the following formula: % specific lysis = [(experimental sample cpm - spontaneous release cpm)/(maximal release cpm - spontaneous release) cpm)] x 100.

Immunohistochemistry

Spinal cords were perfused with PBS, mounted in OCT medium (TissueTek, Elkhart, IN), and frozen in isopropanol cooled in liquid nitrogen. Frozen sections were cut at 6 µm and stored at -70°C until use. Before staining, sections were thawed, air dried for 30 min, and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Following fixation with 4% paraformaldehyde, sections were blocked with normal goat or rabbit serum (2–10%) for 20 min at room temperature before incubation with the primary Abs for 45 min to 1 h at room temperature. The following Abs were used for cellular markers. CD4 (1:100 GK1.5; American Type Culture Collection, Rockville, MD), GFAP (1:1000; Dako), or Fas (1:500 M-20; Santa Cruz Biotechnology, Santa Cruz, CA). The ABC-AP Elite rapid staining system (Vector Laboratories, Burlingame, CA) was utilized according to manufacturer’s instructions and developed with either 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (NBT/BCIP) or red substrate (Vector Laboratories).

Apoptotic cell staining

TUNEL staining was performed to demonstrate apoptotic cells in the spinal cords using the in situ apoptosis kit (Boehringer Mannheim, Indianapolis, IN). After fixing, sections were blocked with 3% hydrogen peroxide for 10 min at room temperature and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. A fluorescein-tagged TdT catalyzed reaction allowed for the detection of apoptosis-induced 3' OH DNA-strand breaks. An anti-fluorescein Ab-peroxidase conjugate was then used to detect the apoptotic cells using 3,3'-diaminobenzidine as the substrate (Vector Laboratories) and visualized using light microscopy. For double-labeling experiments, cellular markers were first performed followed by TUNEL staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma}-treated astrocytes can present all the predominant Theiler’s virus epitopes to T cell clones and hybridomas

Using splenocytes as APC, we and others have previously determined that the regions VP1233–250, VP274–86, and VP324–37 of TMEV represent the major epitopes recognized by CD4+ T cells from the highly susceptible SJL/J strain of mice (10, 39, 40). To determine whether astrocytes, a CNS resident cell population, are able to present the same viral epitopes, we utilized a panel of TMEV-specific, CD4+ T cell clones and hybridomas that recognize the predominant viral determinants (Table IGo). Fig. 1Go demonstrates the levels of IL-2 produced by various TMEV-specific T cell hybridomas and clones in response to UV-inactivated virus or peptides presented by splenocytes and IFN-{gamma}-treated or untreated astrocytes. Splenocytes and astrocytes pretreated with IFN-{gamma} were able to efficiently process and present these viral epitopes to specific T cell hybridomas (Fig. 1GoA). Although untreated astrocytes were not generally able to present to the T cell hybridomas, two hybridoma clones (4D1 and B7) produced low but detectable levels of IL-2. Interestingly, some of the hybridomas, with unknown specificity and VP1-reactive in particular, were stimulated more efficiently by IFN-{gamma}-treated astrocytes than splenocytes. In addition, IFN-{gamma}-treated astrocytes could also present TMEV or viral peptides to T cell clones derived from the CNS of TMEV-infected mice (Fig. 1GoB). These data strongly suggest that astrocytes have the potential to process and present all the predominant T cell determinants of TMEV, similar to or better than APC in the periphery.


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  		Table I. Fine epitope specificity of TMEV-specific T cell hybridomas and T cell clones utilized in this study

 


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  		FIGURE 1. IFN-{gamma}-treated astrocytes process TMEV and the epitopes to various virus-specific T cell hybridomas and T cell clones. Bars represent the level of IL-2 production on stimulation of various T cell hybridoma clones (1 x 105/well) cultured in triplicate with 12.5 µg/ml of UV-BeAn in the presence of 5 x 105 irradiated splenocytes, 6 x 103 untreated (solid bars), or IFN-{gamma}-treated (shaded bars) astrocytes. PBS was used as a negative control. Astrocytes were cultured with UV-TMEV alone, without T cell hybridomas (none), as an additional control. Results are expressed as the incorporation of [3H]TdR by an IL-2-dependent cell line, CTLL-2, grown in the presence of Ag-stimulated hybridoma culture supernatants and are illustrated as {Delta} cpm (mean cpm from UV-TMEV-stimulated cultures - mean cpm from PBS-stimulated cultures). A, IL-2 production by various T cell hybridomas reactive to the predominant viral epitopes upon stimulation with UV-inactivated TMEV in the presence of splenocytes or astrocytes as APC. B, IL-2 production by representative T cell hybridomas and clones upon stimulation with UV-TMEV or epitope-bearing peptides (VP1233–250 or VP274–86 for the corresponding T cells) in the presence of splenocytes or astrocytes.

 
Astrocytes interfere with the proliferative response of T cell clones but not IFN-{gamma} production

Prostaglandins released by cultured astrocytes are known to inhibit cellular immune functions (41, 42). Initially, epitope-induced proliferation by a VP1-specific T cell clone was assessed in the presence of various doses of indomethacin, a potent inhibitor of prostaglandin synthesis. As shown in Fig. 2Go, at 1 x 104 astrocytes, as much as 91% of the proliferative response was inhibited in the absence of indomethacin. However, at 5 x 103 astrocytes, the proliferative response was not significantly affected by prostaglandins released by the astrocytes as the levels of T cell proliferation were not significantly different from each other in the presence or absence of indomethacin.



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  		FIGURE 2. Effect of prostaglandins on astrocyte-stimulated proliferation and IFN-{gamma} production by a representative TMEV-specific T cell clone. A, T cell clone TV-3.11 was cultured with 1 µM VP1233–250 peptide for 4 days in the presence of various concentrations of the prostaglandin inhibitor, indomethacin (see inserted box), and various numbers of IFN-{gamma}-treated astrocytes as a source of APC (x-axis). HEL34–45 peptide was used as a negative control. Results are expressed as {Delta} cpm ± SEM (mean cpm from VP1233–250-stimulated cultures - mean cpm from HEL34–45-stimulated cultures) from triplicate cultures. B, After 42 h, supernatant was removed from the above cultures and assayed for the presence of IFN-{gamma} by ELISA. Results are expressed as the mean ng/ml ± SD of IFN-{gamma} from VP1233–250-stimulated cultures - mean ng/ml of IFN-{gamma} from HEL34–45-stimulated cultures.

 
To examine the relationship between the inhibition of T cell proliferation by prostaglandins and the production of proinflammatory cytokine(s), we have determined the level of IFN-{gamma} produced by the T cells in cultures containing various numbers of astrocytes. It is particularly important to assess the level of IFN-{gamma} because this proinflammatory cytokine is known to play a key role in the immune-mediated inflammatory response leading to the pathogenesis of demyelination. Interestingly, at a concentration of astrocytes (1 x 104) capable of inhibiting the proliferation of a virus-specific T cell clone, the production of IFN-{gamma} by the T cell clone was unaffected (Fig. 2GoB), i.e., 16 and 15 ng/ml of IFN-{gamma} were produced in the presence or absence of indomethacin, respectively. Thus, astrocytes may suppress the proliferation of virus-specific T cells, without affecting the production of IFN-{gamma} (Fig. 2GoB), an important proinflammatory cytokine involved in the immune-mediated inflammatory demyelination.

Activated astrocytes induce proliferative responses of various TMEV-specific T cell clones as well as bulk T cells from virus-infected mice in the presence of indomethacin

To assess whether IFN-{gamma}-activated astrocytes are also able to induce proliferative responses of T cells specific for the predominant viral epitopes, CNS-derived T cell clones specific for VP1233–250 and VP274–86 were stimulated with UV-inactivated TMEV in the presence of either astrocytes or splenocytes as APCs (Fig. 3GoA). Three different patterns for activated astrocytes to process/present TMEV were noted when compared with that of splenocytes. First, activated astrocytes are much more efficient in presenting viral epitopes to induce proliferative responses of VP1-specific (TVF.1) and VP2-specific (TV 6.9) clones in the presence of 2 µg/ml indomethacin. Second, the efficiencies of epitope presentation between astrocytes and splenocytes are similar for another VP2-specific clone (TV-7). Third, splenocytes are much more efficient than activated astrocytes in stimulating VP1-specific TV-3.11. These patterns of stimulatory efficiency were very reproducible with the same T cell clones. Thus, the differences in the stimulatory efficiencies appear to be dependent on the individual T cell clones and not associated with a particular epitope.



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  		FIGURE 3. IFN-{gamma}-treated astrocytes present TMEV epitopes to virus-specific T cell clones and bulk splenic T cells from virus-infected animals. A, TMEV-specific T cell clones (3 x 104/well) were stimulated with various concentrations of UV-TMEV for 4 days in the presence of 6 x 103 untreated (open symbols) or IFN-{gamma}-treated (closed symbols) astrocytes. PBS was used as a negative control. In addition, indomethacin (2 µg/ml) was added to the cultures. B, Nylon wool-enriched splenic T cells (6 x 105/well) from intracerebrally (1 x 106 PFU) TMEV-infected SJL/J mice were incubated with either normal irradiated syngeneic splenocytes (5 x 105/well) or IFN-{gamma}-treated astrocytes (1 x 104/well) with 5 µg/ml indomethacin in the presence of UV-TMEV or antigenic peptides (1 or 10 µM). Results are expressed as {Delta} cpm ± SEM from triplicate cultures.

 
To determine the ability of astrocytes to present viral Ags to bulk T cell populations, nylon wool-separated splenic T cells from TMEV-infected SJL/J mice were similarly stimulated with either normal splenocytes or activated astrocytes as APCs in the presence of UV-inactivated TMEV or peptides bearing individual epitopes (Fig. 3GoB). Although the levels of T cell stimulation were consistently higher with splenic APCs, activated astrocytes were also able to stimulate the splenic T cells with either TMEV or individual epitope peptides. This result appears to be somewhat different from the result in Fig. 3GoA, in that many T cell clones derived from demyelinating CNS lesions are more efficiently stimulated by activated astrocytes. T cell clones from the CNS may include preferentially expanded populations by the CNS resident APCs. Taken together, these results indicate the ability of IFN-{gamma}-activated astrocytes to present all the predominant viral epitopes to virus-specific T cell hybridomas, spinal cord-derived clones, and bulk T cells from infected animals with varying degrees of efficiency.

Virus-specific CD4+ T cells mediate cytolysis of IFN-{gamma}-treated astrocytes

Activation of certain CD4+ T cells results in the cytolysis of target cells (43). To determine the consequences of T cell activation via Ag presentation by astrocytes, virus-specific CD4+ T cell clones derived from the CNS of TMEV-infected mice were cultured with IFN-{gamma}-activated astrocytes pulsed with epitope peptides. Fig. 4Go demonstrates specific lysis of astrocytes by two representative Th1 cell clones, TV-3.11 (VP1233–250-specific) and TV-6.9 (VP274–86-specific). The T cell clones lysed IFN-{gamma}-treated astrocytes in an epitope-specific manner, i.e., VP1-specific T cell clone lysed astrocytes pulsed with VP1233–250, but not with VP274–86, and vice versa for the VP2-specific clone. Pretreatment of astrocytes with IFN-{gamma} appears to be necessary for this T cell-mediated lysis, hence T cell clones were not able to lyse unactivated astrocytes pulsed with specific epitope-peptides (data not shown). Treatment of the cultures with anti-IAs Ab inhibited lysis of peptide-pulsed, activated astrocytes (data not shown), demonstrating that this CD4+ T cell-mediated lysis is MHC class II-dependent. Furthermore, a high level of prostaglandin does not appear to block such T cell-mediated lysis, since the addition of a prostaglandin inhibitor, indomethacin, did not affect this class II-dependent target cell lysis (data not shown). Thus, these results indicate that virus-specific CD4+ T cells have the potential to lyse activated astrocytes. Interestingly, Th2 clones recognizing the wild-type VP1233–250 epitope or an altered VP1233–250 found in the low-pathogenic variant virus (44) did not display similar cytolytic ability. Taken together, these results strongly suggest that inflammatory Th1 cells have a more efficient cytolytic function for activated astrocytes.



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  		FIGURE 4. Lysis of IFN-{gamma}-treated astrocytes by TMEV-specific CD4+ T cell clones. 51Cr-labeled, IFN-{gamma}-treated astrocytes (~1 x 104/well) were pulsed with 1 µM concentrations of the indicated peptides before adding various E:T ratios of the representative T cell clones, TV-3.11 or TV-6.9. Results are expressed as the percent specific lysis ± SEM from triplicate cultures. T cell-mediated astrocyte lysis in the absence of peptide (PBS) resulted in the same low percent lysis as in the presence of negative control peptides (data not shown).

 
Activated astrocytes are killed in vitro by spinal cord cell suspensions from TMEV-infected mice

To examine the possibility that such class II-restricted CD4+ T cells capable of lysing IFN-{gamma}-activated astrocytes are detectable in the CNS of virus-infected mice, the ex vivo lytic activity of T cells in the spinal cords of infected mice was compared with that of uninfected control mice. Single-cell suspensions of spinal cord homogenates from TMEV-infected mice, but not uninfected control mice, were able to lyse IFN-{gamma}-treated astrocytes pulsed with peptides containing the major Th epitopes of TMEV (Fig. 5Go). Spinal cord cells from TMEV-infected mice demonstrated a significantly higher level of specific lysis (up to 46%) of astrocytes pulsed with the viral epitopes but not a nonspecific peptide, HEL (<3%). In contrast, such cells from uninfected control mice resulted in very low lysis (<5%) of astrocytes, regardless of the peptides used. These results strongly suggest that infiltrating Th lymphocytes in the CNS of infected mice are able to lyse astrocytes pulsed with class II-restricted viral epitope peptides.



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  		FIGURE 5. Lysis of IFN-{gamma}-treated astrocytes by spinal cord cell suspensions from virus-infected mice. Astrocytes (5 x 104/well) were incubated in IFN-{gamma} (100 U/ml) for 72 h before labeling with 51Cr. The cultures were pulsed with VP1233–250 (1 µM), VP272–86 (1 µM), and VP324–37 (10 µM) peptides or a nonspecific peptide HEL34–47 (1 µM) as control, for 30 min before the addition of the spinal cord cell suspensions from virus-infected and uninfected SJL/J mice. Spinal cords from two to three mice were pooled to prepare Histopaque-purified single-cell suspensions.

 
Apoptosis of activated astrocytes is mediated by virus-specific Th cells via a Fas-FasL interaction

CD4+ T cells activated by either Con A or anti-CD3 display two distinct mechanisms of cytotoxicity (45), i.e., Fas-FasL/TNF-{alpha} or perforin-mediated. To understand the mechanism by which CD4+ T cells lyse IFN-{gamma}-activated astrocytes, anti-TNF-{alpha}, Fas-Fc serum from transgenic mice, or control serum were used in the cytolytic assays described above. Fig. 6Go demonstrates that anti-TNF-{alpha} Ab was not able to inhibit the specific lysis mediated by a virus-specific Th1 cell clone. However, Fas-Fc was able to completely inhibit the specific lysis induced by the T cell clone (Fig. 6Go). Interestingly, IFN-{gamma}-activated astrocytes displayed a high level of Fas, as well as FasL, on the surface (data not shown). These results indicate that virus-specific Th cells can mediate their cytotoxicity toward IFN-{gamma}-activated astrocytes via a Fas-FasL-dependent mechanism.



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  		FIGURE 6. Inhibition of lysis of 51Cr-labeled, IFN-{gamma}-treated astrocytes by CD4+ T cell clones by Fas-Fc but not anti-TNF-{alpha} Ab. Astrocytes labeled with 51Cr and pulsed with specific epitope peptide were subjected to a standard chromium release assay in the presence of control serum (2%), Fas-Fc serum, or anti-TNF-{alpha} (1 mg/ml). Only Fas-Fc was able to completely inhibit the cytolysis of IFN-{gamma}-treated astrocytes by a representative T cell clone (TV 3.11).

 
Astrocytes undergoing apoptosis as well as astrogliosis are detected in the CNS of virus-infected mice

Immunohistochemical studies were conducted using double staining for apoptosis by TUNEL and astrocytes by anti-GFAP Ab (Fig. 7Go). A drastic increase in TUNEL+ astrocytes was observed in the white matter of the spinal cords of TMEV-infected mice compared with uninfected control mice. In addition, a significant increase in the number of hypertrophic fibrillary astrocytes was found in the spinal cord of virus-infected mice (Fig. 7GoB). Thus, despite the high level of astrocytes undergoing apoptosis, the overall cell number may be increased due to active astrogliosis as observed in the lesions of MS patients (46). Although numerous TUNEL+ cells are GFAP+, many are not, indicating that other cell types, such as oligodendrocytes, may also undergo apoptosis in the spinal cord during demyelination as was previously shown (47). On the other hand, TUNEL+ CD4+ T cells were found to be minimal in the inflammatory lesion when similarly examined by double staining with TUNEL and anti-CD4 Ab (Fig. 7GoE). These results strongly suggest that astrocytes are important resident APCs in the CNS and their subsequent apoptosis may contribute to the pathogenesis of virally induced, immune-mediated demyelination.



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  		FIGURE 7. A significant increase of apoptotic astrocytes in the spinal cords of TMEV-infected mice. In situ detection of apoptotic cells by TUNEL combined with cellular staining using specific Abs (GFAP for astrocytes and CD4 for T cells) were performed to assess the level of apoptosis and the target cell types undergoing apoptosis in the spinal cords following TMEV infection. At least three mice per experiment group were tested for immunohistochemistry, and representative sections are presented. Light micrographs showing the white matter of spinal cords double stained with GFAP (red) and TUNEL (brown) (A-C) or CD4 (blue) and TUNEL (brown) (D–E). A, A section from a control mouse showing only GFAP staining and no TUNEL staining. B, A section from a TMEV-infected mouse (120 days postinfection) showing numerous double-stained cells (GFAP+ astrocytes and TUNEL). Note that a significant number of astrocytes are TUNEL-positive in the lesion. In addition, a significant level of astrogliosis is seen in the white matter of the spinal cord from virus-infected mice. No staining is seen outside of the astrogliotic area, as shown on the bottom right of the section. C, At higher magnification (x600), an example of an astrocyte undergoing apoptosis is shown. D, A section from a control mouse showing neither CD4+ nor TUNEL+ staining indicating the lack or very low level of CD4+ T cell infiltration. E, A section from a TMEV-infected mouse (120 days postinfection) showing numerous CD4+ cells (blue), but only very few are double-stained with TUNEL. A higher magnification (x400) is shown in F. Arrow points to an area with numerous TUNEL-positive cells. Bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of tissue-specific cell populations to process and present foreign Ags to infiltrating lymphocytes may be of critical importance in the development of localized tissue-specific, inflammatory immune responses. During inflammatory conditions of the CNS, it is believed that pathogenic T lymphocytes migrate across the blood-brain barrier and interact with resident neuroglial cell populations capable of presenting specific Ags to infiltrating T cells (48). Therefore, the ability of neuroglia to process and present pathogenic T cell determinants would be a critical step in the initiation of T cell-mediated pathology in the CNS. The expression of MHC Ags on certain neuroglial cell populations during inflammatory conditions (28, 29, 31), and the ability of these cells to process and present Ags in vitro (25), supports the potential role of these cells in Ag presentation in the CNS. A previous finding (24) that class II expression on astrocytes from resistant animals is not readily inducible following IFN-{gamma} treatment compared with susceptible animals in TMEV-induced demyelination is consistent with this possibility.

Here, we have specifically examined the role of astrocytes as an APC population in a CD4+ T cell-mediated, virus-induced demyelinating disease model. It was previously observed that MHC class II gene products are expressed on astrocytes in spinal cord sections from susceptible strains of mice infected with TMEV (18, 49). In addition, IFN-{gamma}-treated astrocytes were shown to present undefined epitopes of Theiler’s virus to T cells from virus-immunized mice (24). However, very little is known about the potential differences in the function of astrocytes as APC compared with peripheral APCs. Utilizing the predominant Th cell determinants of TMEV, we have demonstrated that IFN-{gamma}-activated astrocytes can process TMEV and present all the major epitopes to virus-specific T cell hybridomas, T cell clones, as well as bulk T lymphocytes from virus-infected mice ( Figs. 1–3GoGoGo). Interestingly, some T cell clones derived from the CNS are much more efficiently stimulated by activated astrocytes (Fig. 1Go), although the significance of this preferential T cell activation is not yet clear. Nevertheless, these results indicate that the processing and presentation of viral determinants by astrocytes is at least as efficient as splenocytes. Therefore, the epitopes originally defined using "professional" APC populations from the periphery (i.e., splenocytes) are also likely generated by such "nonprofessional" APC population within the CNS microenvironment.

The consequence of T cell activation within the CNS by resident neuroglia remains a controversial issue. Various reports have demonstrated that astrocytes can suppress cellular immune functions through the production of soluble inhibitors, such as prostaglandins (41, 42). In the EAE model, it has been demonstrated in situ that T cells lose their proliferative ability after infiltrating the CNS (50), suggesting that the inhibitory role of astrocytes on cellular responses in vitro may also have important implications in vivo. Our results examining astrocyte-mediated viral Ag presentation to various T cells indicate that only a narrow range of astrocyte numbers result in T cell proliferation in the absence of a prostaglandin inhibitor, indomethacin (Fig. 2Go). Interestingly, at a concentration of astrocytes (1 x 104/well) in which T cell proliferation is almost completely inhibited, the production of IFN-{gamma} by the T lymphocytes was not affected. Therefore, while T cell proliferation is inhibited by prostaglandins produced by activated astrocytes, those T cells may still produce inflammatory cytokines promoting immune-mediated pathogenesis. However, previous studies using peripheral APCs reported that purified prostaglandin E2 preferentially inhibits the production of Th1-type cytokines, including IFN-{gamma}, over Th2 cytokines (51, 52). This discrepancy may reflect differences in the type of APC or concentration and/or type of prostaglandins produced by astrocytes. Much elevated levels of Th1 as well as Th2 cytokines are found in the CNS of mice with TMEV-IDD (53) supporting this possibility. Therefore, activated astrocytes may function differently from those peripheral APCs, with respect to stimulation of T cells to proliferate and produce proinflammatory cytokines (e.g., IFN-{gamma}). These data may have important implications in vivo, since it has been suggested that the local production of proinflammatory cytokines by activated, virus-specific T lymphocytes is involved in the immune-mediated destruction of the myelin sheath (54).

Another potential consequence of T cell activation by astrocytes is the MHC class II-restricted lysis of these APCs by virus-specific, CD4+ Th lymphocytes. Th1 clones can mediate strong cytolysis utilizing the Fas-FasL mechanism and Th2 cells weakly cytolytic involving perforin-mediated mechanisms (45, 55). Our preliminary experiments showing the inability of Th2 T cell clones to lyse astrocytes correspond to the failure of the Th2 response to exacerbate this Th1-mediated disease (56). Cytolysis of astrocytes and oligodendrocytes by CD4+ T cells have been reported (37), and such mechanism has been proposed to contribute to pathogenesis in EAE (57). However, the type of cells undergoing apoptosis during immune-mediated demyelination is somewhat controversial. Oligodendrocyte apoptosis has been mainly observed in the spinal cords of mice infected with TMEV DA strain (47). In contrast, studies with EAE show the apoptosis of effector T cells and microglia (58, 59). While significant apoptosis of oligodendrocytes and microglia are not excluded, our results strongly indicate a relatively high level (30–60%) of Fas/FasL-mediated apoptosis for astrocytes, as compared with CD4+ T cells. (Fig. 7Go). As far as we are aware, this is the first report documenting such apoptotic astrocytes in the demyelinating lesions. It has recently been demonstrated that Fas/FasL-mediated apoptosis of T cells is not dependent on the engagement of TCR apparently involved in maintaining immune-privileged sites, e.g., in the eye and testis (60). However, Fas-FasL apoptosis of target cells mediated by CD4+ T cells apparently requires the engagement of TCR via peptide-loaded class II molecules as shown here and by others (57). Thus, the elimination of Ag-presenting resident cells is much more discrete as compared with the infiltrating T lymphocytes in the CNS, an immune privileged site. This mechanism may be involved in the effective removal of specific cells that are infected with virus or potentially harmful by inducing inflammatory responses leading to the development of autoimmunity (61). However, the destruction of Ag-presenting astrocytes may compromise the blood-brain barrier, promoting inflammatory demyelination.

Virus-specific, CD4+ Th1 cell clones derived from the CNS or bulk T lymphocytes from the spinal cords of TMEV-infected mice appear to lyse the epitope-presenting astrocytes via Fas-FasL interaction (Fig. 4Go). This is consistent with previous studies suggesting the role of Fas-FasL interaction in multiple sclerosis (62) and similarly in autoimmune mouse models of demyelination (63, 64, 65). Therefore, it is conceivable that astrocytes play a critical role for viral Ag presentation to T lymphocytes and, subsequently, become a potential target for the T cells. Since astrocytes are responsible for maintaining the highly impermeable blood-brain barrier (66), the Fas-FasL-mediated cytotoxicity by Th1 cells may compromise the integrity of the blood-brain barrier. Furthermore, such injury of astrocytes may lead to vigorous fibrillary astrogliosis attempting to repair the initial damage. Similar astrogliosis has been observed in human MS (46) and EAE with increased astrocyte activation due to proinflammatory cytokines (67). Fibrillary astrogliosis may result in the ill-organized, abnormal support for the endothelial cell layer maintaining the blood-brain barrier. In addition, activated astrocytes are known to produce chemokines attracting various inflammatory cells (67). These may promote the influx of inflammatory cells into the CNS further exacerbating immune-mediated demyelination. However, the role of Fas-FasL-mediated apoptosis of activated astrocytes by stimulated T cells in the pathogenesis of virally induced demyelination may be very complex and dependent on the level of T cell infiltration in the CNS, virus-induced apoptosis, and viral persistence.


   Acknowledgments
 
We thank Ron Koehler, Ziad Alnadjim, Dr. Terrence Barrett, and Dr. Mauro Dal Canto for their help in the immunohistochemical studies; and Dr. Shyr-Te Ju for the generous gift of Fas-Fc serum.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants RO1 NS28752, RO1 NS33008, and RO1 NS23349. J.P.P. was supported by National Multiple Sclerosis Society Postdoctoral Fellowship FG1172-A-1. Back

2 Current address: Sugen, Inc., Cell Survival, 230 East Grand Avenue, South San Francisco, CA 94080. Back

3 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: Back

4 Abbreviations used in this paper: MS, multiple sclerosis; TMEV, Theiler’s murine encephalomyelitis virus; TMEV-IDD, TMEV-induced demyelinating disease; EAE, experimental allergic encephalitis; GFAP, glial fibrillary acidic protein. Back

Received for publication November 11, 1998. Accepted for publication March 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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