c substrain differences in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology52 (1994) 19-24

BALB/c substrain differences in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease S i m o n e M . N i c h o l s o n a, J e f f r e y D . P e t e r s o n a S t e p h e n D . M i l l e r M a u r o C. D a l C a n t o b, R o g e r W . M e l v o l d ,,a

a, K e g i a n g

Wang

a,

a Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Acenue, Chicago, IL 60611, USA b Departments of Pathology and Neurology, Northwestern University Medical School 303 E. ChicagoAL'enue, Chicago, IL 60611, USA

Received 11 October 1993;revision received and accepted 10 February 1994

Abstract

We report differences among BALB/c substrains in susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, an immune-mediated inflammatory demyelinating disease and experimental model for human multiple sclerosis. BALB/cJ and BALB/cAnNCr mice are susceptible, while BALB/cByJ and BALB/cCum are resistant. Hybrids between BALB/cBy and BALB/cAnNCr were intermediate, although closer to the resistant parent. Backcrosses gave results compatible with differential susceptibility being related to a single segregating locus. Exposure of resistant BALB/cByJ mice to low dose irradiation, 2 days prior to infection, rendered them susceptible to TMEV-induced demyelination. The susceptibility pattern of TMEV-induced demyelinating disease among BALB/c substrains is distinct from those of several autoimmune disorders. Key words: Theiler's murine encephalomyelitis virus; BALB/c; Demyelinating disease; Multiple sclerosis; Autoimmunity

1. Introduction

B A L B / c is one of the most widely utilized strains of inbred mice. As a result of separation and isolation, various B A L B / c substrains differ at some loci, a few of which have been specifically identified, and in their susceptibility to the development of several immunemediated diseases, including experimental allergic orchitis or EAO (Teuscher et al., 1985a,b, 1987a,b, 1988), experimental allergic encephalomyelitis or E A E (Munoz and Mackay, 1984; Hickey et al., 1986; Teuscher et al., 1987b, 1988), and multi-system autoimmune diseases after infection with encephalomyocarditis virus (EMCV) (Babu et aI., 1985). We report here differences among four B A L B / c substrains for susceptibility to a vitally induced inflammatory disease of the CNS, Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD), a model for human multiple

* Corresponding author. Phone (312) 503 4105; Fax (312) 503 1339 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-5728(94)00026-K

sclerosis. At least one of the resistant substrains becomes susceptible when exposed to 200 rads of y irradiation prior to infection. The pattern of genetically d e t e r m i n e d T M E V - I D D susceptibility/resistance among B A L B / c substrains is quite distinct from those found for EAE, EAO and EMCV-related autoimmune disease in other studies, although one of the other diseases, EAE, also serves as an experimental model for human multiple sclerosis.

2. Materials and methods 2.1. A n i m a l s and inoculations

Mice of the B A L B / c B y J and B A L B / c J substrains were purchased from the Jackson Laboratory. Mice of the B A L B / c A n N C r substrain were purchased from the National Cancer Institute. At 5 - 7 weeks of age, mice were anesthetized with methoxyfluorane and inoculated in the right cerebral hemisphere with 2.9 × 106 PFU of BeAn 8386 virus. All animals were examined

S.M. Nicholson et al. /Journal of Neuroimmunolog3' 52 (1994) 19--24

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twice weekly for the first 60 days, and weekly thereafter up to 90-120 days post-inoculation, for development of clinical neurological symptoms, particularly the characteristic chronic gait abnormality caused by demyelination. Control mice were uninjected, or (in some cases) injected with media alone or with BHK lysate, but were kept in the same environment as the experimental animals.

2.4. Irradiation Mice were irradiated in a Gammacell 40 Small Animal Irradiator (Cs 137 source), at approximately 123 rads per rain, for a total exposure of 200 rads. The dual source arrangement ensures equal irradiation to all specimens.

2.2. Clinical signs

2.5. Histopathology

Animals were graded (blindly) for the characteristic waddling gait associated with TMEV-induced demyelinating disease, and each was assigned a clinical score based on the following criteria: 0, no abnormality in gait; 0.25, any slight abnormality in gait not severe enough to be associated with demyelination; 1, swaying gait; 2, full waddling gait and spastic paralysis. Animals with scores of 1 or 2 were considered to be affected.

At 105-120 days post-inoculation, mice from each strain were anesthetized and killed by perfusion through the left ventricle with 10 ml of PBS (pH 7.3), followed by 100 ml of chilled 3% glutaraldehyde in phosphate buffer (pH 7.3). Spinal cords were dissected from the vertebral canal, sectioned at approximately l-ram intervals, post-fixed in 1% osmic acid for 1 h and processed for Epon embedding. 10-/xm sections from each spinal cord were stained with toluidine blue and examined by light microscopy. Pathological changes were graded as follows: - (no disease); _+ (meningeal inflammation); + (focal parenchymal inflammation with demyelination); + + (multiple areas of parenchymal inflammation and demyelination); + + + (extensive inflammation and demyelination with confluent lesions).

2.3. Virus The BeAn 8386 strain of T M E V was isolated from a feral mouse in Belem, Brazil in 1957 and was later classified as a T M E V by complement fixation serology (Rohzon et al., 1983). After plaque purification and titer amplification by serial passage in BHK-21 cells, a working stock was prepared with a titer of 9.7 × l0 s P F U / m l , which was subsequently diluted in D M E by a factor of 10 for inoculation (30 /xl administered per mouse). As controls for the effect of other infectious agents in the environment, uninfected animals (receiving i.c. inoculations of either D M E or BHK-21 cell lysate, but no T M E V ) were maintained together with the TMEV-infected animals.

2.6. Statistics The INSTAT statistical program (GraphPAD Software, Inc.) was used to compare the frequency of disease in different substrains with Fisher's Exact Test (two-tailed) for 2 × 2 contingency tables.

Table 1 Differential susceptibility to T M E V - I D D among B A L B / c substrains Group

A

Substrain

TMEV b

NO. affected/ No. tested

BALB/cJ

+

-

0/5 (0%)

C

BALB/cAnNCr

+

23/33 (70%)

D E

BALB/cByJ

+

0 / 1 8 (0%) 6 / 4 2 (13%)

F G

BALB/cCum ~

+

0 / 1 8 (0%) 0 / 1 0 (0%)

B

8 / 1 0 (80%)

Inflammation c

50% + 50% +

Significant diff. ( P values) vs. J

AnNCr

ByJ

Cum

N/A

n.s.

0.0001

0.0004

N/A

0.0001

0.001

N/A

n.s.

50% + 50% + + > 80% < 20% +_ ND d

N/A

Data from Clatch et al. (1987). The B A L B / c C u m mice were obtained from Cumberland View Farms. All other procedures (e.g. virus strain and dose, age at inoculation) were as described in Materials and methods. b Uninfected controls received i.c. inoculations of either D M E or BHK-21 lysate, but no TMEV. c Grading scale described in Materials and methods. d Not done in this study; refer to Clatch et al. (1987).

S.M. Nicholson et al. /Journal of Neuroimmunology 52 (1994) 19-24

21

Table 2 Genetic and induced susceptibility differences between the ByJ and A n N C r substrains Group

Substrain

Irradiation

Affected/total

A B C

F1 hybrids a (ByJ X AnNCr)F1 x ByJ (ByJ x AnNCr)F1 x A n N C r

N/A N/A N/A

15/50 (30%) 9 / 2 9 (31%) 6/11 (55%)

D E

BALB/c AnNCr BALB/cAnNCr

None 200 r

2 4 / 3 2 (75%) 13/15 (87%)

P =

F G

BALB/cByJ BALB/cByJ

None 200 r

4 / 3 6 (11%) 3 4 / 3 9 (87%)

P = 0.00001

a

Probability

n.s,

No difference observed between reciprocal hybrids: (ByJ X AnNCr; 5 / 1 8 ) and ( A n N C r X ByJ; 10/32).

2. 7. Tail skin grafting Split thickness tail skin grafts were reciprocally exchanged between anesthetized mice and observed regularly for 90 days. The tail skin grafting method has been previously described in detail (Bailey and Usama, 1960; Kohn and Melvold, 1975).

3. Results

The differential susceptibilities to TMEV-IDD of three B A L B / c substrains currently studied (BALB/cJ, BALB/cByJ, BALB/cAnNCr) and one (BALB/ cCum) from a previous report (Clatch et al., 1987) are shown in Table 1. The clinical scores are based on the characteristic gait abnormality associated demyelination, and were confirmed by histopathology on samples from each group. B A L B / c J (Group A) and B A L B / cAnNCr (Group C) are quite susceptible to the disease (80% and 70% susceptible, respectively), while B A L B / cByJ (Group E) and BALB/cCum (Groups G) are relatively resistant (13% and 0% susceptible, respectively). The 13% seen for BALB/cByJ mice is elevated over our usual experience. All six of the affected animals occurred in a single experiment, one of the few

times we have seen them display clinical symptoms. The susceptibility of the B A L B / c A n N C r and BALB/cJ substrains has been typical in our experience. The affected B A L B / c animals usually develop only a mild gait abnormality and are more comparable, with respect to severity of clinical symptoms, to those strains classified as intermediately susceptible (e.g. A / J , C3H) than to those which develop severe disease (e.g. DBA/2, SJL/J, SWR/J, PL/J). Control animals (Table 1; Groups B, D and F), maintained with the experimental groups but not infected with TMEV, showed no clinical symptoms of demyelination. Representative affected and unaffected animals were examined histologically for the presence of demyelinating lesions. The presence of characteristic demyelinating lesions, with mononuclear cell infiltration, correlated with clinical signs. Among the more susceptible of the substrains, BALB/cJ showed meningeal inflammation with about half of the sections also displaying focal parenchymal lesions (+ to + ) and BALB/cAnNCr mice showed mild to moderate parenchymal lesions with about half of the sections graded as + and about half as + + (Table 1; refer to grading system in Materials and Methods). The mild to moderate inflammation was concordant with the intermediate severity of the clinical symptoms.

Table 3 Lack of correlation of T M E V - I D D susceptibility with previously reported B A L B / c substrain differences

a

Substrain

Differential genotypes

J ByJ AnNCr Cum O R N L

f

Disease susceptibilities TMEV-IDD

EAE b

EAO c

EMCV d

Afr-1 e

Qa-2 c

susc res susc res

res susc susc -

res susc susc -

susc res

b a a a (?)

a b b -

-

susc

susc

-

-

-

a EAO, experimental allergic orchitis; EMCV, encephalomyocarditis virus; Afr-1, ~-fetoprotein regulation (chrom. 15; alleles a and b); Qa-2, Qa lymphocyte antigen 2 (chrom. 17; alleles a and b); res, resistant; susc, susceptible; - , not known. b Munoz and Mackay (1984); Hickey et al. (1986); Teuscher et al. (1987b, 1988). c Teuscher et al. (t985a,b, 1987a,b). d Babu et al. (1985). e Lyons and Searle (1990); these four sublines do not differ at Gdc-1, although some other B A L B / c substrains do. f B A L B / c O R N L (Oak Ridge National Laboratory) is the substrain from which B A L B / c C u m was subsequently derived.

22

S.M. Nicholson et aL /Journal of Neuroimmunology 52 (1994) 19 24

Hybrids between the susceptible AnNCr and resistant ByJ substrains (Table 2) showed a slightly greater incidence of disease (30%) than did the resistant ByJ substrain (13%), although the increase was not statistically significant (P > 0.07). The F1 hybrids were, however, significantly less susceptible than were the AnNCr animals (P = 0.005). There was no maternal effect as hybrids with ByJ mothers showed the same incidence of disease (5/18) as did hybrids with AnNCr mothers (10/32). Thus, resistance appears to be the dominant phenotype. Backcrosses of the hybrids to both parental substrains gave results compatible with the differential susceptibility being due to a single segregating locus (Table 2), although the data is insufficient to exclude the involvement of additional loci. Backcrosses to the dominant resistant ByJ parental substrain showed about the same degree of susceptibility (31%) as seen in the hybrids (30%). Backcrosses to the recessive susceptible AnNCr parent displayed an increase in susceptibility (55%) above that of the hybrids, but the number of animals (6/11) is low due to animals lost due to a husbandry mishap. If a single locus were involved, the backcross population should be composed of roughly equal number of heterozygotes and recessive homozygotes for the locus in question. If the disease frequency is 30% in the former (from Table 2) and 70% in the latter (from Table 1), the overall susceptibility in the backcross animals would be expected to be about 50%, which is compatible with the 6/11 observed. The low number of backcross animals in this combination, however, needs to be expanded in future studies for more discriminating analyses.

3.1. Effects of irradiation on the resistant ByJ substrain It has been previously reported, in the experimental autoimmune encephalomyelitis (EAE) model for MS, that resistant BALB/c substrains can be converted to the susceptible phenotype after exposure to 200 rads of 3' irradiation prior to infection (Lando et al., 1980). Similar phenomena have been reported in the TMEV system for C57BL/10 mice (Rodriguez et al., 1990) and for B6D2F1 mice (Olsberg et al., 1993; Pelka et al., 1993). Exposure to 200 rads of whole body y irradiation, 2 days prior to infection with TMEV, resulted in an increase of disease incidence among BALB/ByJ mice to 87% (Table 2), a frequency comparable to that normally seen in the susceptible BALB/cAnNCr mice. Irradiation did not increase disease incidence in susceptible BALB/cAnNCr mice.

and therefore suitable for future experiments involving adoptive transfers, split thickness tail skin grafts were exchanged between the substrains. All grafts were accepted (12/12 from ByJ donors to AnNCr recipients; 9,/9 from AnNCr donors to ByJ recipients; grafts were observed for 55 days), indicating that there are no significant histocompatibility barriers between the two substrains.

4. Discussion

TMEV is a natural enteric pathogen of wild and laboratory mice. Upon access to the central nervous system by either occasional spontaneous means or by deliberate inoculation, TMEV displays a general viremia before clearance from the brain at about 2-3 weeks. TMEV subsequently establishes a low level, persistent infection in the white matter which stimulates an inflammatory, virus-specific immune response, resulting eventually in immune-mediated demyelination in the white matter, 1-2 months post-infection. Some strains display high susceptibility to TMEV-IDD, some display intermediate susceptibility and others are highly resistant (Lipton and Dal Canto, 1979). BALB/c has traditionally been listed among the resistant strains. We first noted exceptions in 1989, when we were forced to switch from BALB/cByJ to BALB/cAnNCr because of the fire which destroyed part of the Jackson Laboratory facilities. The unexpected results with BALB/cAnNCr suggested subline diversity similar to that reported for other autoimmune and infectious diseases (Table 3), prompting the present study. The original substitution of BALB/cAnNCr for BALB/cByJ was made, in part, because the two substrains are quite closely related, having diverged in 1961. However, as shown in Fig. 1, the susceptibility/resistance pattern does not correlate with the genealogical pattern (Potter, 1985; personal LINEAGE OF RELEVANT BALB/c SUBSTRAINS

1937

1961

1937-38

1959

3.2. Histocompatibility testing of the AnNCr and ByJ substrains In order to determine whether the susceptible AnNCr and resistant ByJ substrains were histocompatible,

ByJ AnNCr J ORNL Cum Fig. 1. Genealogy of the BALB/cByJ, BALB/cJ, BALB/cAnNCr and BALB/cCum substrains

S.M. Nicholson et aL /Journal of Neuroimmunology 52 (1994) 19-24

communication from Dr. K.E. Kile, Sr. of Cumberland View Farms). The more resistant substrains (ByJ and Cure) were derived independently from the more susceptible substrains. It may be that the genetic difference(s) between BALB/cByJ and B A L B / c A n N C r which affect susceptibility to TMEV-IDD are different from that between B A L B / c C u m and BALB/cJ. Additionally, the susceptibility/resistance pattern among B A L B / c substrains does not correlate with either of the two loci at which the substrains used are known to differ (Afr-1 and Qa-2;, see Table 3). B A L B / c J a n d / o r BALB/cByJ have been previously utilized for studies of EMCV-induced multisystern autoimmune disease (Babu et al., 1985), experimental allergic orchitis (EAO) and experimental autoimmune encephalomyelitis (EAE). The pattern for susceptibility to TMEV-IDD is distinct from those described for the other diseases (Table 3). EAO is an autoimmune inflammation of the male reproductive organs, after immunization with testicular antigens (Teuscher et al., 1985a,b, 1987a,b, 1988). Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammation of the central nervous system, after immunization with components of myelin (Munoz and Mackay, 1984; Hickey et al., 1986; Teuscher et al., 1987b, 1988). E A E and T M E V - I D D result in immune-mediated demyelination of the white matter of the CNS and both serve as models for human multiple sclerosis, although the immune responses are directed against different antigenic specificities in the two diseases. EAE results from immune responses directed at 'self' antigens of myelin such as myelin basic protein (MBP) and proteolipoprotein (PLP). TMEV-IDD, in contrast, results from responses directed specifically against components of the virus itself. Anti-TMEV T cell-mediated immune responses generated in infected mice, however, do not appear to be cross-reactive with myelin components (Miller et al., 1987, 1989). Thus, unlike EAE, EAO and EMCV-related multi-system disease, the preponderance of the evidence indicates that TMEV-IDD is not autoimmune in nature. In addition, B A L B / c mice (substrain unspecified), normally resistant to development of EAE, can be made susceptible by either treating them with cyclophosphamide or irradiating them prior to immunization with MBP (Lando et al., 1979, 1980). Analyses of differential susceptibility to TMEV-IDD among different inbred mouse strains have thus far identified the involvement of several distinct loci including H-2D (one of the class I loci of the mouse major histocompatibility complex) on chromosome 17 (Rodriguez et al., 1986; Melvold et al., 1987a); Tmevd-1 ( T M E V demyelination-1) on chromosome 6 (Melvold et al., 1987b), and Tmevd-2 on chromosome 3 (Melvold et al., 1990). Bureau et al. (1992) have also identified loci affecting TMEV viral persistence which include H-2D

23

and more than one non-H-2 loci. Other, as yet unmapped, loci are also likely to be important, at least in some strain comparisons. No specific gene differences, however, have thus far been identified between B A L B / c A n N C r and BALB/cByJ. In previous strains or strain combinations, where pretreatment with irradiation or cyclophosphamide has been shown to convert resistant animals to the susceptible phenotype, it has also been shown that resistance can be restored by the adoptive transfer of spleen cells from non-pretreated, syngeneic donors (Lando et al., 1979; Olsberg et al., 1993; Pelka et al., 1993). However, the question of whether this 'induced' susceptibility (and its reversal) parallels the genetically determined differences between susceptible and resistant strains remains unclear, because comparable cell transfers between genetically disparate animals face strong histoincompatibility barriers, even where only single locus differences are involved (Rodriguez et al., 1986; Melvold et al., 1987a). The availability of a set of closely related and histocompatible B A L B / c substrains, in which both induced and genetically determined differences in TMEV susceptibility exist, should provide a unique opportunity to determine whether the differences/mechanisms in 'induced' susceptibility are the same as those dictated genetically. Experiments are currently in progress to see, for example, whether adoptive transfers of splenic T cells from unirradiated resistant C / B y J mice can 'protect' both irradiated, infected C / B y J and normally susceptible C / A n N C r mice. If so, and if the effective splenic cell subset is the same in both cases, it would suggest that the 'induced' form of susceptibility indeed provides a good model of genetically determined susceptibility.

Acknowledgements We thank Shuyi Wu, Amy Pelka, Bansi Joshi and Janie Tong-Kokkinakis for their assistance, and Colleen Olsberg for helpful discussions. This work was supported in part by Grants NS-23349 (R.W.M.) and NS13011 (M.D.C.) from the USPHS.

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