Abstract Theiler's murine encephalomyelitis viruses. (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain ...
Acta Neuropathol (1996) 91 : 595–602
© Springer-Verlag 1996
R E G U L A R PA P E R
Ikuo Tsunoda · Yuzo Iwasaki · Hiroshi Terunuma · Kazuya Sako · Yoshiro Ohara
A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus
Received: 26 June 1995 / Revised, accepted: 27 December 1995
Abstract Theiler’s murine encephalomyelitis viruses (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain produces acute polioencephalomyelitis in mice, whereas the DA strain produces demyelination with virus persistence in the spinal cord. A comparative study of GDVII and DA strains suggested that low host immune responses are responsible for the development of acute GDVII infection and that the persistence of infected macrophages plays a crucial role in the development of chronic white matter lesions in DA infection. All 78 mice infected with GDVII died or became moribund by day 13, while none of 54 mice infected with DA died. In the acute stage, the distribution of viral antigens in the central nervous system (CNS) tissue was similar in both GDVII and DA infections, although the virus titer was higher in GDVII infection. In DA infection, a substantial number of T cells were recruited to the CNS on day 6 when they were virtually absent in GDVII infection. The titer of neutralizing antibody was already high on day 6 in DA infection but was negligible in GDVII infection. Development of chronic paralytic disease from day 35 of the DA infection was accompanied by focal accumulation of viral antigen-positive macrophages in the spinal white matter. In addition, white matter lesions comparable to those in chronic DA infection were induced in the spinal cord within 7 days af-
I. Tsunoda1 · Y. Iwasaki · H. Terunuma · K. Sako · Y. Ohara Department of Neurological Sciences, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980, Japan Tel.: 81-22-273-9105; Fax: 81-22-274-3847 Y. Iwasaki (Y) Miyagi National Hospital, Yamamoto-cho, Watari-gun, Miyagi 989-22, Japan Tel.: 81-223-37-1131; Fax: 81-223-37-3585 Present address: 1 Department of Neurology, University of Utah, School of Medicine, Medical Center, 50 No. Medical Drive, Salt Lake City, UT 84132, USA
ter intracerebral injection of DA-infected murine macrophages. Key words Murine encephalomyelitis virus · Demyelinating diseases · Inflammation · Macrophages
Introduction Theiler’s murine encephalomyelitis viruses (TMEV) belong to the Picornaviridae, and are divided into two subgroups, GDVII and DA, on the basis of their biological activities. The GDVII strain causes acute fatal polioencephalomyelitis in mice [19, 22], whereas the DA strain induces a biphasic disease [18]: flaccid paralysis developing several days after virus inoculation and demyelination of the spinal cord occurring 4–5 weeks later. This demyelinating disease is thought to be an experimental model for multiple sclerosis [25, 36]. Although recent studies on chimeric recombinants of these subgroup viruses have partly clarified the genes or gene products responsible for biological differences [7, 8, 33], the precise mechanisms governing these biological activities are still not fully understood. In DA infection, the involvement of immunopathological mechanisms has been postulated for demyelination. A depletion of T cells in vivo has been shown to reduce the severity of demyelination in chronic infection [20, 28, 35] and Gerety et al. [9, 10] identified a VP2 epitope responsible for the generation of immunopathological T cells. While phenotypic analysis of mononuclear cell (MNC) infiltrates may provide a clue to elucidate the pathomechanism of tissue damage in inflammatory disease [13], in TMEV infection, only the phenotype of cellular infiltrates induced by the DA strain has been analyzed [3, 4, 17] and no comparative studies between the two subgroups have been reported. The association of virus persistence in macrophages with chronic white matter degeneration is recognized in a variety of retrovirus infections including HIV infection [14]. DA strain antigens and viral RNA have been found
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to persist in macrophages in chronic DA infection [2, 6, 29]. More recently, Lipton et al. [21] found that the predominant virus antigen burden resides within macrophages in chronic DA infection. It has also been reported that DA virus persistently infects brain macrophages in vitro [11, 16] and infectious virus has been recovered only from a macrophage-enriched fraction of central nervous system (CNS) tissue in chronic DA infection [5]. These findings suggest a close association of macrophage infection with chronic white matter disease, but little is known about the role of the infected macrophages in the development of such disease. To elucidate the role of host immune responses in TMEV infections, we correlated the virus titer, localization of virus antigens and histopathological changes with the antibody titer, phenotypes of MNC infiltrates in the CNS and lymphoproliferative response to TMEV of spleen cells in GDVII and DA infections. Furthermore, to understand the interaction between TMEV and host macrophages, the cytotropism of the two strains to a murine macrophage cell line was studied as a possible mechanism to discriminate the subgroups with regard to demyelinating activity. In addition, white matter lesions were induced in the spinal cord within 7 days after inoculation of a DA-infected macrophage cell line.
Materials and methods Propagation and purification of virus GDVII and DA viruses were initially propagated in baby hamster kidney (BHK-21) cell cultures maintained in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum. Virus titer was determined by a plaque assay in L-929 cells [26]. Original stocks of GDVII and DA had an infectivity of 9.0 × 108 plaque-forming units (PFU)/ml and 2.5 × 108 PFU/ml, respectively. To enhance the neurovirulence [15], DA virus was further subjected to nine serial passages in suckling mouse brains. Tissue homogenate (20% in MEM) (brain-DA) prepared from the final passage had a titer of 1.3 × 105 PFU/ml. Virus antigens for in vitro lymphoproliferative assay were prepared as described previously [30, 31]. Animal experiments Three-week-old C3H/He male mice purchased from the Funabashi Farm (Japan) were used unless otherwise stated. Mice under ether anesthesia were intracerebrally inoculated with 20 µl of either GDVII or brain-DA virus with an infectivity of 0.9 × 105 PFU/ml and 1.3 × 105 PFU/ml, respectively. The culture medium containing 1% fetal bovine serum was used as a control inoculum for GDVII and a 20% normal mouse brain homogenate in MEM for brain-DA. Virus titer was assessed by a plaque assay and an antibody titer was determined by the 50% plaque-reduction test using L-929 cells [26]. Flow cytometry On days 4 and 6 for GDVII and days 6, 11, and 35 for brain-DA virus infection, 15 mice of each subgroup were killed by exsanguination under ether anesthesia. MNC in the blood samples were separated by centrifugation through a Ficoll-Paque gradient (Pharmacia). MNC in the CNS tissue were separated on discontinuous
Percoll gradients [5]. Brain and spinal cord tissues were removed en bloc and were dissociated by passing them through a stainless steel screen. The dissociated tissues were suspended in PBS and pelleted at 400 g for 10 min. Each suspension was overlaid with 5 ml of 30% Percoll, and centrifuged at 1,200 g for 30 min at 20°C. MNC were recovered from the 30%/68% Percoll interface. At each time point, five pools of cell suspensions, each from three mice, were subjected to flow cytometry. The following mouse monoclonal antibodies (mAb) were employed: fluoresceinisothiocyanate (FITC)-conjugated anti-CD3, anti-CD4 (antiL3T4), and anti-CD8a (anti-Ly-2) from Caltag; FITC-conjugated anti-mouse IgG and a rat mAb F4/80 for mature mouse macrophage from Serotec. As a second antibody for F4/80, phycoerythrin-conjugated anti-rat IgG (Caltag) was used. Approximately 5 × 105 MNC were reacted with mAb for 45 min at 4°C and appropriately diluted for flow cytometry using a FACScan (Becton Dickinson). For the F4/80 stain, the cells were further incubated with the secondary antibody for 30 min at 4°C. Negative controls were incubated with PBS alone in place of the first antibody or with an irrelevant mAb of the same isotype. A total of 5,000– 10,000 cells were scanned for each fraction. Data were analyzed with a Consort 30 computer program. Immunohistochemistry Mice were perfused with 10% buffered formalin under ether anesthesia. Brains were divided into six coronal slabs and spinal cords in ten slabs which were embedded in paraffin by standard methods. Sections (3 µm thick) were prepared and were immunostained by the avidin-biotin complex (ABC) as described previously [12]. Monocyte/macrophage lineage cells of cellular infiltrates and microglia were identified by RCA-1 lectin staining. Although vascular endothelial cells were also faintly visualized with RCA-1, they could be distinguished from infiltrating macrophages and activated microglia based on their morphology and localization [34]. Viral antigens were detected with an anti-GDVII mAb (GDVIImAb1) from Dr. R. P. Roos, University of Chicago Medical Center. This mAb reacts with VP1 epitope of both TMEV subgroups [24]. Double-immunofluorescence staining for macrophage and viral antigens was carried out using the biotinylated RCA-1 combined with fluorescein avidin D (Vector) and GDVII mAb1 combined with a rhodamine-conjugated goat anti-mouse IgG (Tago). Negative controls for the staining protocol consisted of omitting the primary antibody or RCA-1. Adjacent sections were stained with hematoxylin and eosin, and Luxol fast blue. TMEV lymphoproliferative assay The lymphoproliferative response of spleen cells to TMEV [4] was assayed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric method [23]. The optimal dose of viral antigen for this assay was found to be 1 µg in a preliminary experiment. Viable spleen cells (2.5 × 105) were cultured in the presence or absence of UV-inactivated purified virus in 0.1 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum in 96-well flat-bottom plates. The cultures were incubated for 96 h. Then 10 µl of MTT (5 mg/ml) solution (Chemicon) was added to each well and the cells were incubated for another 4 h. The plates were read with a MTP-32 microplate reader (Corona electric) using a test wavelength of 575 nm and reference wavelength of 630 nm. The cultures were prepared in triplicate and the results were expressed as stimulation indices (SI) = [mean optical density (OD)575–630 of stimulated cultures]/[mean OD575–630 of control cultures]. TMEV infection in a murine macrophage culture The murine macrophage cell line J774.1 [27] was obtained from the Cancer Cell Repository, Tohoku University. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal
597 bovine serum. The cells were infected with either GDVII or DA virus grown in BHK-21 cells at a multiplicity of infection (MOI) of 1 PFU/cell. After virus adsorption, the cells were washed and refed with RPMI 1640 containing 1% fetal bovine serum. The culture supernatants and cell lysates were separately titrated by plaque titration on L929 cells. Inoculation of TMEV-infected macrophages J774.1 cells (H-2d) were infected with DA virus grown in BHK-21 cells at an MOI of 0.1 PFU/cell and harvested at 12 h post-infection (DA-J774 cells). Under these conditions, approximately 5% of DA-J774 cells in these cultures were TMEV antigen positive. Three-week-old susceptible SJL/J (H-2s), intermediately susceptible C3H/He (H-2k), and resistant BALB/c (H-2d) mice were used. Under ether anesthesia, the mice were inoculated intracerebrally with 1 × 106 DA-J774 cells (2 × 105 PFU). As a control, mice were inoculated with 1 × 106 uninfected J774.1 cells. Animals were killed on days 3, 7, 14, 21, and 28 after infection. For virus titration, the cerebral hemisphere opposite to the side of virus inoculation and the spinal cord were used. Portions of the brain and spinal cord were processed for light microscopic observations.
a
Results Clinical disease, virus titer and antigen distribution Most GDVII-infected mice developed sluggish movements, trembling and a hunched back on day 4, and all of 78 infected mice died or became moribund between days 4 and 13. The virus titer rose to 1.5 × 106 PFU on day 4 and 1.4 × 107 PFU on day 6 (0.6 g CNS tissue) (Fig. 1a). In the GDVII-infected mice, viral antigens were readily detected in the neuronal perikarya, dendrites and axons on day 4. Groups of antigen-positive neurons were seen in the neocortex, the entorhinal cortex, the hippocampus, the septal nuclei, the thalamus, the hypothalamus, the periaqueductal gray matter, the pontine tegmentum, the deep cerebellar nuclei and the spinal gray matter. On day 6, dying neurons with chromatin condensation were widely scattered throughout the brain and the spinal cord (Fig. 2a), and the number of antigen-positive neurons increased (Fig. 2b). Inflammatory cell reaction was inconspicuous but RCA-1-positive MNC were present in the parenchyma. On the other hand, none of 54 mice infected with brain-DA died but 17 mice developed hindlimb paralysis between days 8 and 12. The paralysis, however, improved in 14 out of 17 mice within a week; 3 remained paraplegic. Between days 25 and 30, another 4 mice developed paralysis. Moreover, paralysis was aggravated in 3 mice that had partially recovered from the acute disease. On day 6, 5.8 × 105 PFU of virus was recovered but the titer rapidly declined thereafter (Fig. 1b). In contrast to the GDVII infection, parenchymal as well as perivascular and subarachnoidal MNC infiltrations were conspicuous in brain-DA infection (Fig. 2c). The distribution of viral antigens on day 6 of the brainDA infection (Fig. 2d) was similar to that in GDVII-infected mice but the number was smaller. On day 11, viral antigens were rarely detected in neurons. On day 35, the inflammation in the brain had largely subsided, leaving focal necrotic lesions where viral antigens were rarely
b Fig. 1 Virus titers in the central nervous system (CNS) and serum neutralizing antibody (Ab) titers in GDVII infection (a) and in brain-DA infection (b). Each point represents the average of three mice
seen. Instead, focal demyelinating lesions associated with accumulation of antigen-positive cells were seen in the spinal cord. Although RCA-1-positive macrophages, some of which contained virus antigens, were observed on days 11 and 35 in brain-DA infection, as already described by several authors [2, 6, 21, 29], we could not detect viral antigens in macrophages in GDVII infection. Antibody responses On day 6, the titer of neutralizing antibody was already high (1:512) in brain-DA-infected mice, whereas in GDVII-infected mice, it remained low (1: 16), and many mice died before there was a significant rise in the antibody titer (Fig. 1). In brain-DA infection, the serum antibody titer reached 1:2048 on day 35. Phenotypes of MNC infiltrates in CNS tissue Since a substantial number of MNC were recovered from the mice inoculated with control inoculum [(28.9 ± 5.1) ×
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a
b
c
d
Fig. 2a–d Representative histopathology and antigen distribution on day 6. The hippocampus of the mice infected with GDVII (a, b) and brain-DA (c, d). a, c H&E stain, b, d TMEV antigens by the avidin-biotin-peroxidase complex method. a–d × 94
Table 1 Number of MNC isolated from CNS. Total number of MNC isolated from CNS of C3H/He mice on different days following infection with TMEV. Each group contained five pools composed of specimens from three mice (MNC mononuclear cells, CNS central nervous system, TMEV Theiler’s murine encephalomyelitis virus, PID post-inoculation day)
104cells/mouse on day 6, (16.8 ± 2.3) × 104cells/mouse on day 35], only the increments from these numbers were interpreted as the number of inflammatory cells related to the virus infection. In GDVII infection, despite a rapid increase of virus load in the CNS, the number of MNC recovered from the CNS was smaller than that in brain-DA infection (Table 1). Although F4/80-positive cells were predominant in both GDVII and brain-DA infections, a larger number of CD3 cells were recovered from brain-DA-infected mice on day 6 (Fig. 3). The percentages of CD3 cells in the brain-DA infection were 43.5% on day 11 and 34.7% on day 35 (Table 2). The CD4/CD8 ratio was constantly lower in the CNS (0.8–1.5) than in the peripheral blood (2.5–3.3), suggesting preferential infiltration of CD8 cells into the CNS lesions. While in the peripheral blood, surface immunoglobulin (sIg) was detected in 25–35% of MNC in brain-DA-infected mice and 13% of MNC in GDVII-infected mice, sIg-positive cells were rarely seen in the CNS in either GDVII or brain-DA infections (Table 2).
Virus GDVII DA
PID
No. of MNC/CNSa (× 104 cells/mouse)
pb
4 6
0.7 ± 8.9 15.9 ± 12.2
0.05
6 11 35
26.2 ± 9.4 15.5 ± 5.9 3.7 ± 4.7
0.16 0.05 < 0.01
a Number of MNC isolated from control group was subtracted. Data are mean ± SD b Statistical difference by unpaired Student’s t-test
TMEV lymphoproliferative assay of spleen cells The stimulation indices (SI) of the spleen cells of brainDA-infected mice were 1.9 on day 6 and 2.1 on day 35, respectively. No proliferative response was seen in the spleen cells from GDVII-infected mice on day 6 even after the antigen dose was changed to either 0.1 µg or 5 µg.
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a
Fig. 4 The number of viable cells in uninfected, GDVII and DAinfected J774.1 cell cultures
viruses could establish a highly productive infection in murine macrophages. b
Pathogenicity of DA-infected macrophages in mouse CNS
Fig. 3 Quantitation of mononuclear cells (MNC) isolated from the CNS of the mice infected with GDVII (a) and brain-DA (b). At each time point, five samples pooled from three mice each were subjected to FACScan analysis. *P
