INTRODUCTION

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Theiler's murine encephalomyelitis virus (TMEV) is a member of the family Picornaviridae, genus Cardiovirus, and a natural enteric pathogen of mice. TMEV has been divided into two groups based on neurovirulence following intracerebral inoculation of genetically susceptible mice. Infection with BeAn, a low-neurovirulence strain, results in a chronic, demyelinating disease of the central nervous system (CNS). Persistence of BeAn virus leads to activation of major histocompatibility complex class II-restricted CD4⁺ Th1 lymphocytes directed at virus epitopes (11, 13, 14, 33, 34) and immunopathologic damage of myelin. This animal model has been used as an experimental analogue of multiple sclerosis.

During TMEV persistence, the major virus antigen burden resides in CNS macrophages (Ms) (12, 29, 37); however, only a relatively small percentage of Ms infiltrating demyelinating lesions contain detectable virus antigen (29), and an infected M produces only 1 to 5 PFU (12). To investigate virus-M interactions that might lead to restricted virus replication, we have used the myelomonocytic precursor cell line, M1. Recently, it was reported that the M1 cell line was susceptible to virus infection only after differentiation into M-like cells (M1-D cells) (22). BeAn infection of M1-D cells is highly restricted and results in apoptotic cell death that requires virus replication. In the present study, M1-D cells were treated with gamma interferon (IFN-) prior to BeAn virus infection to determine whether activated Ms secreting antiviral cytokines would inhibit BeAn virus replication and to explain, at least in part, the low virus titers in the spinal cords of infected mice. As expected, BeAn-infected, IFN--activated M1-D cells showed no evidence of virus replication; unforeseen was the observation that activated M1-D cells died by apoptosis.

We describe experiments to define the mechanism(s) by which activated M1-D cells die after BeAn infection. Soluble FasL, tumor necrosis factor alpha (TNF-), and TNF- apoptosis-inducing ligand (TRAIL) induced significant cell death in M1-D cells after IFN- activation. RNase protection assay (RPA) showed increased Fas and TNF-R p55 (p55 chain of the TNF- receptor) mRNA transcript levels with activation alone and increased TRAIL mRNA with activation and infection.

One possible scenario suggested by the data is that IFN- activation sensitizes these cells to death-inducing ligands. Virus infection after activation also causes increased IFN-/ (22) and results in up-regulation of TRAIL and increased secretion of TNF-, both of which result in cell death. Thus, depending on the activation state, BeAn virus induces apoptosis in Ms either through an intrinsic mechanism requiring virus replication or through an extrinsic mechanism involving the death receptors and their ligands.

	MATERIALS AND METHODS

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Cells, viruses, and reagents. M1-D cells were differentiated from the M1 cell line with supernatants from L929 and P388D1 cells as previously described (22) and maintained in complete medium containing 10% fetal bovine serum. Cells were either left untreated or treated with 100 U of IFN- (Sigma, St. Louis, Mo.) per ml for 24 h unless otherwise specified. Soluble TRAIL was a generous gift of James Pan (Genentech, South San Francisco, Calif.), TNF- was purchased from Sigma, and soluble FasL was purchased from Alexis Corporation (San Diego, Calif.). TNF- was analyzed by the mouse TNF- immunoassay kit, Quantikine M, from R&D Systems (Minneapolis, Minn.). The inhibitors soluble TNF- RII (sTNF-RII) and recombinant human TRAIL-R2:Fc (rhTRAIL-R2:Fc) were obtained from Oncogene Research Products (Cambridge, Mass.) and from Alexis Corporation, respectively. The origin and passage history of the BeAn virus stock have been described (39). Virus titers of clarified lysates of infected cells were determined by standard plaque assay on BHK-21 cells (39).

Virus infections. After virus adsorption at a multiplicity of infection of 10, or as indicated, for 45 min at 24°C, M1-D cells were washed with phosphate-buffered saline, pH 7.2, and incubated in complete RPMI medium containing 5% fetal bovine serum at 37°C in a 5% CO₂ atmosphere for the indicated times.

MTT assay. Cell viability was determined by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to blue formazan crystals as previously described (23). Briefly, 10⁴ cells were incubated for 2 to 4 h with 50 µl of MTT (1 mg/ml) 20 to 24 h after infection and/or treatment with cytokines. Formazan crystals were dissolved in 100 µl of dimethyl sulfoxide, and the optical density at 560 nm (OD₅₆₀) was read on a UV Max microplate reader (Molecular Diagnostics, Palo Alto, Calif.). Statistical analysis was performed on quadruplicate samples using Cricket Graph 1.3 unless otherwise indicated.

Flow cytometry. After blocking nonspecific antibody binding with 10% goat serum and/or 2 µl of anti-CD32/16 (FcR) (PharMingen, San Diego, Calif.), cells were incubated with polyclonal rabbit anti-BeAn antiserum (1:1,000), polyclonal rabbit anti-TRAIL antiserum (1:100)(Alexis Corporation), or hamster anti-FasL monoclonal antibody (1:100) (PharMingen). Secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) F(ab')₂ (1:200) (Cappel/Organon Teknikon, Durham, N.C.) and biotinylated anti-hamster IgG cocktail (1:100) (PharMingen). A 1:200 dilution of avidin-FITC (PharMingen) was used as the fluorochrome for the biotinylated antibody. Cytoplasmic antigen was detected as previously described (21). After staining, cells were fixed in 1% paraformaldehyde and analyzed on a FACSCalibur (Becton Dickinson, Palo Alto, Calif.). Data were evaluated using CELLQuest 3.1f supplied with the instrument. Annexin V staining using Annexin V-FITC apoptosis detection kit (Sigma) was performed according to the manufacturer's instructions.

Western blot analysis. Cell lysates were prepared in buffer containing 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 100 µl of protease inhibitor cocktail (Sigma). Cell lysates were clarified by low-speed centrifugation to remove nuclei and debris, and protein content was determined with Bio-Rad DC protein assay kit (Hercules, Calif.) according to the manufacturer's instructions. Samples (40 µg/lane) were electrophoresed on 10 to 12% polyacrylamide gels and transferred to ProBlot membranes (Applied Biosystems, Foster City, Calif.). Membranes were blocked overnight with Tris-buffered saline containing 5% nonfat dry milk and 0.02% Tween 20, washed extensively, and incubated with rabbit polyclonal anti-human TRAIL-R1 or R2 (1:500) (BioSource International, Camarillo, Calif.), anti-human TRAIL (1:1,000) (Alexis Corporation) or anti-caspase 8 (1:100) (Santa Cruz Biotechnology, Santa Cruz, Calif.). After a washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma), washed again, and analyzed by enhanced chemiluminescence (ECL) using SuperSignal West Dura as a substrate (Pierce, Rockford, Ill.).

Assay for viral RNA replication. Viral RNA replication was assayed by incorporation of [³H]uridine in the presence of actinomycin D as previously described (23). Briefly, after virus adsorption, 2 × 10⁴ cells were incubated in a 96-well plate with 100 µl of complete medium containing 5 µg of actinomycin D per ml and 10 µCi of [³H]uridine (ICN; 15 to 25 Ci/mmol) per ml. At the indicated times, samples were harvested with a PHD Cell Harvester (Cambridge Technologies, Watertown, Mass.) and radioactivity was determined using a Beckman scintillation counter (LS5000TD; Palo Alto, Calif.). The mean and standard deviation of quadruplicate samples were calculated using Cricket Graph 1.3.

Immunoprecipitation of virus capsid proteins. Immunoprecipitation of [³⁵S]methionine-labeled virus capsid proteins was as previously described (23). Briefly, 10⁶ cells were incubated with 100 µCi of L-[³⁵S]methionine (ICN; 100 Ci/mmol) for 18 h postinfection (p.i.) and lysed in radioimmunoprecipitation assay buffer. Lysates were clarified by centrifugation, and protein content was determined as described above. After preclearing with normal rabbit serum, BeAn proteins were immunoprecipitated with 5 µl of polyclonal anti-BeAn rabbit antiserum and protein G-coupled Sepharose beads (Sigma). Samples were solubilized in sample buffer and electrophoresed on SDS-polyacrylamide gels.

Assay for caspase activity. Caspase protease activity was measured in cell lysates by release of aminomethylcoumarin from the substrate peptide, acetyl-Asp-Glu-Val-Asp (DEVD), as previously described (22).

RPA. RNA was isolated from M1-D cells using TRIZOL Reagent (Life Technologies, Grand Island, N.Y.) according to the manufacturer's instructions. The RiboQuant Multi-Probe RNase protection assay (RNA) (PharMingen) was used according to the manufacturer's instructions to analyze RNA expression of the bcl-2 family (mAPO-1) and the TNF superfamily members (mAPO-2). Probes were synthesized using [-³⁵S]UTP instead of [-³²P]UTP. Densitometry scans were done on the STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) using ImageQuant version 5.0 software. The numbers were derived as ratios of the housekeeping gene, L32.

	RESULTS

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BeAn virus-induced apoptosis of IFN--activated M1-D cells. While viral RNA replication (Fig. 1A), expression of capsid proteins (Fig. 1B), and production of infectious virus (22) were measurable in M1-D cells, there was no evidence of viral RNA replication after activation with IFN- as measured by [³H]uridine incorporation (Fig. 1A) or of viral protein expression as assayed by immunoprecipitation (Fig. 1B). Mean fluorescence intensities of BeAn virus antigen staining by flow cytometry in M1-D cells and activated M1-D cells were 145 and 18, respectively (Fig. 1C and D) (mean fluorescence intensities for uninfected control cells were 4.6 and 9.7). Although flow cytometry analysis indicated the presence of low levels of viral proteins in activated M1-D cells, this probably represents residual virus and not newly synthesized viral proteins. Plaque assays of lysates from unactivated and activated M1-D cells showed that 2 to 2.5 PFU were produced in each M1-D cell (four samples from two separately differentiated populations done in duplicate) and showed consistent titers of <0.1 PFU for each activated cell. Since activated M1-D cells produce 10-fold higher levels of IFN-/ than do M1-D cells (22), the lack of BeAn replication and protein expression in activated M1-D cells was not surprising (Fig. 1A, B, and D). However, activated M1-D cells showed apoptotic changes after BeAn infection including cell rounding, surface blebbing, and condensed nuclei and loss of adherence (not shown). To confirm the mechanism of virus-induced cell death of activated M1-D cells, caspase activity and annexin V binding were assayed as early indicators of apoptosis (32, 42, 47). Caspase activity increased fivefold in both unactivated and activated infected M1-D cells by 8 h p.i. compared to uninfected controls (Fig. 2A). The kinetics of caspase induction were similar in the two cell populations beginning 4 h p.i., peaking at 8 and 10 h and declining to undetectable levels by 24 h (data not shown). The kinetics of annexin V binding paralleled that of caspase activity, beginning 4 to 6 h p.i. (Fig. 2B). Together these results indicate that both unactivated and activated M1-D cells died by apoptosis after BeAn virus infection (summarized in Table 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Characteristics of BeAn virus infection in M1-D and IFN--activated M1-D cells. (A) BeAn RNA replication in unactivated () or IFN--activated () M1-D cells. Cells were incubated in 100 µl of medium containing 1 µCi of [3H]uridine and 0.5 µg of actinomycin D and harvested at 2-h intervals. Data are presented as means ± standard deviations of 6 samples. (B) Immunoprecipitation of BeAn virus capsid proteins from lysates of [35S]methionine-labeled BeAn-infected unactivated and IFN--activated M1-D cells. One million cell equivalents were lysed in lysis buffer (see Materials and Methods), precleared with normal rabbit serum, and immunoprecipitated with rabbit polyclonal anti-BeAn antiserum. Immunoprecipitated proteins were resolved on a 12% polyacrylamide gel; virus proteins are indicated. The experiment was repeated twice with similar results. (C and D) Representative flow cytometry histograms of permeabilized uninfected (solid line) and infected (dashed line) unactivated (C) and IFN--activated (D) M1-D cells 6 h p.i. stained for BeAn virus antigen.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Evidence for apoptosis in unactivated and IFN--activated M1-D cells. (A) Kinetics of caspase activity in BeAn-infected unactivated () and IFN--activated () M1-D cells and uninfected control cells (). Lysates from 106 cells were analyzed for the ability to cleave the DEVD-aminomethylcoumarin substrate. Data are means of duplicate samples with microtiter plate background values subtracted and are representative of two kinetic experiments. Caspase activity was measured in three other preparations of M1-D cells at 8.5 h p.i. with similar results. (B) Kinetics of annexin V binding to BeAn-infected, unactivated () and activated () M1-D cells. Background values for uninfected cells were 8.2% ± 2.1% and 17.0% ± 6.5%, respectively. Values are representative of two kinetic experiments. (C) MTT assay of cell viability with increasing multiplicities of infection of BeAn virus in unactivated () and activated () M1-D cells. The results shown are representative of four independent experiments. Quadruplicate samples were averaged, and the results are expressed as percentages of uninfected controls. The standard deviation for the OD readings was less than 0.1 for all samples.

View this table: [in this window] [in a new window]   TABLE 1.   Summary of BeAn virus infection in unactivated and IFN--activated M1-D cells

Up-regulation of TRAIL in BeAn-infected, IFN--activated M1-D cells. To determine whether the TNF family of proteins was altered during IFN- activation and/or BeAn virus infection, total RNA was isolated at various times p.i. and analyzed by RPA. mRNA levels of three TNF family members (FAF, TRADD, and RIP) (Fig. 3A) did not change under the conditions tested, and that of FasL was not expressed. Densitometric analysis relative to the housekeeping gene, L32, revealed that caspase 8 and TNF-R p55 mRNA levels decreased with time after infection of unactivated M1-D cells (Fig. 3B). IFN- activation alone up-regulated Fas and TNF-R p55 mRNAs, neither of which changed after infection (Fig. 3C). Caspase 8 mRNA expression increased almost twofold after infection of activated cells, consistent with its role as an initiator caspase in apoptosis (42). TRAIL mRNA was expressed in activated M1-D cells beginning 4 h p.i. and increased 1.5-fold by 8 h (Fig. 3A and C). Neither IFN- activation nor BeAn virus infection alone was sufficient to initiate TRAIL mRNA transcription in unactivated M1-D cells (Fig. 3A and C). Both signals were required to up-regulate TRAIL mRNA, and its transcription correlated with the kinetics of apoptosis (Fig. 2B).

View larger version (44K): [in this window] [in a new window]   FIG. 3.   TRAIL mRNA expression in M1-D cells. (A) RPA of RNA isolated from unactivated and IFN--activated M1-D cells at 2-h intervals after BeAn virus infection. Probes are shown in the far left lane; protected RNA bands are labeled on the right and were identified by size according to manufacturer's instructions. The time course was repeated twice, and samples were subjected to RPA twice with similar results. (B and C) Densitometry scans of RPA illustrated in panel A: caspase 8 (), Fas (), TRAIL (), TNF-R p55 (), L32 housekeeping gene (), and background () in unactivated (B) and IFN--activated (C) M1-D cells.

View larger version (74K): [in this window] [in a new window]   FIG. 4.   Caspase 8 expression in M1-D cells. Lysates from unactivated (A) or IFN--activated (B) M1-D cells at various times after infection are shown. Mouse caspase 8 zymogen is ~50 to 55 kDa, while cleaved products recognized by this antibody are ~40 and 23 kDa as shown. The ECL was exposed for 2 min; the zymogen can be seen in unactivated M1-D cells on longer exposure (20 min); however, background levels are much higher. Cleavage products are seen in the uninfected cell lysates from both cell populations (A and B). More of the procaspase 8 is seen in the IFN--activated M1-D cells than in the unactivated cells.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   TRAIL protein expression in M1-D cells. (A) Flow cytometry of cytoplasmic TRAIL expression in unactivated (light line) and IFN--activated M1-D cells (bold line) 4, 8, and 24 h p.i., respectively. (B) Western blot of unactivated and IFN--activated M1-D cell lysates prepared from samples harvested 4, 8, 12, and 24 h p.i. Polyclonal anti-human TRAIL-R1 (62 kDa) and R2 (62 kDa) (1:500) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:50,000) were used for detection with SuperSignal West Dura substrate by ECL. 3T3 cell lysate was used as a positive control as suggested by the manufacturer.

Effects of sTRAIL, FasL, and TNF- on M1-D cell viability. To determine whether TRAIL receptors were functional in unactivated and activated M1-D cells, soluble TRAIL (sTRAIL) was added to cell cultures and cell viability was analyzed by the MTT assay. Cell death in activated M1-D cells was induced in a dose-dependent manner and averaged 28% (72% viability) at 1 µg of soluble TRAIL per ml (Fig. 6A). Interestingly, unactivated M1-D cells proliferated in the presence of TRAIL, averaging 125% above control values. These results were consistent in two separate experiments using two independently derived M1-D cell populations. The low biological activity of TRAIL in these experiments probably reflects the absence of artificial trimerization, which is required for full biological activity of TRAIL, TNF-, and FasL (5, 45, 46). Nevertheless, TRAIL at higher concentrations significantly decreased cell viability over control cultures in a dose-dependent fashion (Fig. 6A).

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Ability of soluble death ligands to induce apoptosis in unactivated and IFN--activated M1-D cells. (A) Percent viability, measured by the MTT assay, of unactivated () and IFN--activated () M1-D cells treated with increasing amounts of soluble TRAIL. Formazan crystal formation was measured 20 to 22 h after treatment with soluble TRAIL. Quadruplicate samples were averaged, and the results are expressed as percentages of values for uninfected controls (P < 0.02). Results are representative of three independent experiments. (B) Percent viability of unactivated (black columns) and IFN--activated (gray columns) M1-D cells with soluble TRAIL, FasL, and TNF- measured by the MTT assay. Data are presented as means ± standard deviations (SD) of four independent measurements with two different M1-D cell populations. Concentrations are shown. (C) TNF- in supernatants of M1-D cells measured by immunoassay. Supernatants collected at time zero were harvested 24 h after the initial cultures were plated. Symbols: , uninfected M1-D; , BeAn-infected M1-D; , IFN--activated, uninfected M1-D; , IFN--activated, BeAn-infected M1-D. Means of quadruplicate samples and SD are shown.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Inhibition of BeAn virus-induced cell death by TNF-RII and TRAIL-R2. BeAn-infected unactivated (A) or IFN--activated (B) M1-D cells were incubated with increasing concentrations of either sTNF-RII (open bars) or rhTRAIL-R2:Fc (gray bars), and cell viability was assayed by MTT 20 h p.i. Results are shown as mean OD560 and standard deviations of quadruplicate samples. The inhibitors protected IFN--activated M1-D cells, whereas no protection occurred in the unactivated M1-D cells. This experiment was repeated with similar trends.

	DISCUSSION

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Apoptosis can be triggered by a number of stimuli, including viral infections (36) and cell stress (19) (intrinsic pathway), and cell surface signaling through the death receptors and their ligands (TNF-, FasL, and TRAIL) (5) (extrinsic pathway). Our data indicate that both pathways are utilized in BeAn-infected Ms depending on their activation state. Although poliovirus proteins 3C (7) and 2A (15) induce apoptosis directly, the mechanism of apoptosis induction by picornavirus infections has not been clearly defined. Using a yeast two-hybrid system, Henke et al. (20) demonstrated binding of coxsackievirus B3 VP2 to the proapoptotic siva protein; however, TMEV VP2 showed no such binding. Agol et al. (2, 3) described two competing death programs initiated with poliovirus infection: one dependent on caspases to initiate the apoptosis program and the other independent of caspases and resulting in cell death without the hallmarks of apoptosis (canonical cytopathic effect).

Our data show that BeAn virus induces caspase 3-dependent apoptosis in both unactivated and activated M1-D cells. However, BeAn-induced apoptosis in activated M1-D cells is not accompanied by virus replication, whereas apoptosis in unactivated cells requires virus replication. In addition, caspase 8 (an initiator caspase) mRNA expression increases with infection of activated cells, whereas it appears to be degraded in unactivated cells (Fig. 3A and C). The results of Western blotting for caspase 8 protein indicate that the protein is cleaved prior to infection in both unactivated and activated M1-D cells. Caspase 8, as well as the other 13 caspases, resides in the cytoplasm as inactive zymogens, which are cleaved upon activation to initiate the apoptosis program (19). Recent evidence has shown that caspase 8 is cleaved in activated T cells and may be involved in proliferation (4, 27). Los et al. (30) have postulated that caspase 8 may be involved in cell-cycle progression, another checkpoint assuring that only healthy cells progress through the cell cycle. Our data suggest that the caspase 8 proenzyme is already cleaved in M1-D cells. Thus, since M1-D cells have been differentiated in vitro, caspase 8 in these cells may be activated and function in the control of the cell cycle. A more detailed analysis of the caspase family members in M1-D as well as the precursor M1 cells will be required to resolve this issue. Nonetheless, it appears that activated M1-D cells have more of the unprocessed protein present than the unactivated cells, which supports the findings of the RPA.

Since IFN- activation sensitizes death receptors to signaling from their ligands (Fig. 4B), a phenomenon termed activation-induced cell death (24), and BeAn virus infection stimulates production of the ligands, a logical explanation for these data is that M1-D cells generate signals for their own demise. The only source of death-inducing ligands in this system is the activated M1-D cells themselves. Two candidate ligands for apoptosis induction in BeAn-infected, activated M1-D cells are TRAIL and TNF-. After infection, TRAIL mRNA and protein expression are up-regulated and high levels of TNF- are found in the supernatant (Fig. 3 and 6C, respectively). Thus, apoptosis through either ligand is possible. Inhibition of cell death by both sTNF-RII and rhTRAIL-R2 indicates that both molecules are used in this system. These data for cell death receptor and ligand expression are summarized in Table 2.

Two cell death-signaling pathways have been described: an intrinsic pathway due to cellular stress which is dependent on mitochondrial release of cytochrome c and caspase 9 activation and controlled through the Bcl-2 family (17) and an extrinsic pathway which is dependent on ligand binding to death receptors, recruitment of FADD/Mort adaptor molecules, and caspase 8 activation (38). A recent report by Walczak et al. (44) describes experiments in which overexpression of Bcl-2 had no effect on TRAIL-induced apoptosis and expression of caspase 8 was necessary and sufficient for TRAIL-induced apoptosis. Sprick et al. (41) also reported that FADD/Mort1 and caspase 8 are essential for TRAIL-induced apoptosis. Additional evidence that the death receptors and their ligands are involved in BeAn virus-induced apoptosis in activated M1-D cells is that Bcl-2 overexpression had no effect either on caspase 3 activation or on cell viability (unpublished). RPA analysis for seven bcl-2 family members (bcl-W, bfl1, bcl-X, bak, bax, bcl-2, and bad) showed no change in expression with time after infection (unpublished). Therefore, in activated M1-D cells the intrinsic mitochondria-dependent apoptosis mechanism is not used and apoptosis occurs through the extrinsic pathway. Confirmation of this hypothesis awaits detailed analysis of the caspase cascade in these cells.

Recently, various investigators have reported TRAIL up-regulation after infection with several viruses including human cytomegalovirus in fibroblasts (40), measles virus in human monocyte-derived dendritic cells, monocytes, and CD3-activated T cells (43), and reovirus infection in fibroblasts (10). To our knowledge, this is the first report to show that a picornavirus up-regulates TRAIL in an activated M-like cell line and broadens the range of viruses affecting the TRAIL apoptosis pathway.

It was recently reported that human monocyte-derived Ms expressed TRAIL after IFN- and IFN- treatment but resisted TRAIL-mediated apoptosis (18). IFN- and IFN- by themselves did not up-regulate TRAIL expression in our system, but did sensitize M1-D cells to signaling from their death receptors. Additional signals, provided by BeAn virus infection, were required to upregulate TRAIL. Whether this discrepancy is due to species differences, use of primary versus tumor cells, or the different methods of M differentiation induction is unclear.

IFNs (type 1 and 2) induce double-stranded RNA-dependent protein kinase (PKR), which mediates apoptosis (16). To assess the role of PKR in our system, we obtained undifferentiated M1 cells expressing a PKR mutation (p686 [6, 35]); however, we were unable to differentiate these cells in vitro (unpublished data). Infection of mouse peritoneal Ms and CNS microglia from normal mice and mice with the PKR mutation could provide further information on apoptosis-signaling mechanisms in Ms at different stages of activation. Until recently, we have been unable to infect peritoneal Ms with BeAn virus; however, we are now examining the role of TRAIL, TNF-, and Fas in apoptosis induction by infection of these cells. Continuous up-regulation of IFN- and TNF- in the CNS has been reported during the course of TMEV-induced demyelinating disease (8), which may play a role in the immunopathology of the disease. Whether TRAIL also contributes to the immunopathology induced by TMEV is unknown but now merits further study.