Clin. exp. Immunol. (1978) 31, 276-280.

Swan mice: autoimmune mice without xenotropic type C virus production CHRISTINE BLAINEAU, SYLVIE GISSLEBRECHT, DOMINIQUE PIATIER,* MARIEANTOINETTE HUROT & J. P. LEVY Laboratoire d'Immunologie et de Virologie des Tumeurs, INSERM U152, and *Institut de Rhumatologie, Centre de Recherches sur les Maladies Osteo-Articulaires, INSERM U5, Hdpital Cochin, Paris, France

(Received 28 April 1977)

SUMMARY

Swan mice are characterized by various autoimmune disorders and the Swan disease resembles that of (NZB x NZW)F1 hybrids. A virological study of these animals shows that: (a) they do not produce xenotropic type C viruses at a detectable level; (b) old Swan mice can produce ecotropic type C virus but at a low level; and (c) this virus production and the appearance of antinuclear antibodies are dissociated in some mice. These results do not support the role of xenotropic type C viruses in murine autoimmune pathology. A possible role of ecotropic type C viruses cannot be definitely excluded, but appears unlikely.

INTRODUCTION NZB mice and (NZB x NZW)F, hybrids are remarkable for various autoimmune abnormalities. Since the continuous production of a xenotropic type C virus at a high level in all organs from birth to death is a unique feature of NZB mice (Levy et al., 1975b), one could suppose that this virus may be responsible for the autoimmune disorders. It could also possibly account for the appearance of autoimmunity in the (NZB x NZW)Fj hybrids, since xenotropic virus is found in their spleen and kidneys (Levy et al., 1975b). The observation that the gp 69/71 of type C virus and the corresponding antibodies contribute to the immune deposits in the nephritic kidneys of NZB and (NZB x NZW)F1 could support this hypothesis (Yoshiki et al., 1974). The role of a xenotropic agent as the primary cause of the autoimmune disease is, however, still not established. It is therefore interesting to look at other autoimmune mice, providing further models to study the relationship between autoimmunity and type C virus production. We report here the results of a virological study in 'Swan' mice (Monier & Robert, 1974). These mice are characterized by various autoimmune disorders including, like (NZB x NZW)F1 hybrids (Burnet & Holmes, 1965), very high titres of antinuclear antibody (ANA) early in life. By 8 months of life 80-100% of these mice are positive for ANA and the anti-DNA nature of the antibodies has been established (Monier & Sepetjian, 1975). Immunoglobulin deposits are seen in the skin and kidneys in a large number of Swan mice (Monier & Sepetjian, 1975). Moreover, early in life the Swan mice develop autoantibodies reacting with T lymphocytes (Monier & Robert, 1974) and they also show functional alterations of the thymus, including a decreased response of spleen cells to PHA (Monier & Robert, 1974), a poor response to thymus-dependent antigens such as sheep red blood cells (Monier & Robert, 1974) and a rapid fall in the level of 'thymic factor' (Dardenne et al., 1974). On the whole, the autoimmune disease of Swan mice resembles more that of(NZB x NZW)F1 mice than the NZB disease. The expression of both xenotropic and ecotropic type C viruses was looked for in Swan mice and the results show that, despite the autoimmune abnormalities, this strain does not express these agents at an especially high level. Correspondence: Professor J. P. Levy, Laboratoire d'Immunologie et de Virologie des Tumeurs, INSERM U152, Hopital Cochin, 27 rue de Fauborg Saint Jacques, 75014 Paris, France. 0099-9104/78/0200-0276S02.00 (©1978 Blackwell Scientific Publications

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MATERIALS AND METHODS Mice. The Swan mice were obtained from a colony bred at the Hospital Cochin (Paris) from the original Swan stock kindly given by J. C. Monier (Universite Claude Bernard, Lyon). NZB, AKR, C3H and C57BI/6 mice were obtained from our own colony. Titration of antinuclear antibodies. Sera obtained from tail-vein blood were diluted from 1/10 to 1/500 and assessed for the presence of ANA by indirect immunofluorescence on rat liver sections, using fluorescein-conjugated goat antimouse IgG or IgM sera (Meloy Laboratories, Maryland). Cells. The CCL 64 mink cell line, which is highly sensitive to xenotropic murine leukaemia viruses (MuLV) (Henderson, Lieber & Todaro, 1974) and to amphotropic type C viruses (Hartley & Rowe, 1976), was obtained from Dr B. Guillemain (Bordeaux). Ha-NZB cells, which produce a xenotropic pseudotype of the Harvey sarcoma virus (Levy et al., 1975b), and normal rat kidney cells were kindly provided by Dr J. Harvey (London). BALB/3T3 (Aaronson & Todaro, 1968), C3H cells (Reznikoff, Brankow & Heidelberg, 1973) and XC cells (Svoboda, 1961) were a gift of Dr B. Bassin (Bethesda, U.S.A.). Xenotropic type C virus (X-MuLV) assays. Xenotropic type C viruses are defined as those growing in xenogeneic cells at the exclusion of any mouse cell (Levy, 1973). The assays were performed in 6-19 months old female animals, since the ANA titres increase with age and are generally higher in females than in males (Monier & Sepetjian, 1975), whereas the X-MuLV replication is apparently sex-independent in NZB mice (Levy et al., 1975b). Spleens were aseptically removed and a single cell suspension was prepared. Usually 1/10 of the spleen cells of individual mice were co-cultivated with mink cells. After 4-6 weeks of subcultures, virus replication was tested by two different assays: (a) competition in the MuLV p30 radioimmunoassay, using acellular extracts prepared from the cultured mink cells, as described by Parks et al. (1973); and (b) the presence of exogenous reverse transcriptase activity in supernatants, determined according to the method of Gerwin & Milstein (1972). This latter test was done under the supervision of J. C. Cherman (Institut Pasteur, Paris). Xenotropic MuLV neutralization. Advantage was taken of the existence of the Ha-NZB cell line, which produces a xenotropic pseudotype of Harvey sarcoma virus (xenotropic Ha-SV) (Levy et al., 1975b). Half log-diluted sera of Swan mice were incubated for 30 min at room temperature with xenotropic Ha-SV and the mixtures were used to infect NRK cells. Clearly detectable foci of transformed cells were counted 6-7 days following infection. The titre of neutralization is expressed as the reciprocal of the highest serum dilution which reduces by 50% the number of foci in control cultures. Detection of ecotropic MuLV. The ecotropic type C viruses, in contradiction with xenotropic MuLV, replicate in murine cells. They are defined as N tropic, B tropic or NB tropic, according to their ability to replicate in NIH (N), BALB/c (B) or both (NB) (Hartley, Rowe & Huebner, 1970). They were assayed by the splenocyte plaque assay described by Melief et al. (1975). Serial dilutions of individual spleen cell suspensions were used to infect monolayers of sensitive indicator mouse cells of two types: C3H cells (Reznikoffet al., 1973) (N type) and BALB/3T3 (Aaronson & Todaro, 1968), or C57BI/6 embryonic fibroblasts (B type). On day 5 the cell monolayers were irradiated with u.v. in 106 XC cells immediately added to each plate. On day 7, the plates were fixed and stained with Giemsa.

RESULTS Study of the X-MuLV production in Swan mice The co-cultivation experiments revealed that none of the thirteen Swan mice tested, the ANA titres ofwhich varied from 1/10 to. 1/250, produced a detectable level of X-MuLV. Considering that we regularly detect high levels of X-MuLV following the infection of mink cells with xenotropic virus (AT 124, NZB cells supernatant) or with lymphoid cells from NZB mice, the possibility that a low level of X-MuLV production could have been missed in these experiments appears very unlikely. Presence of an X-MuLV neutralizing factor in Swan mice It is well-established that a non-immunoglobulinic factor that specifically neutralizes X-MuLV is present in the sera of most, if not all, inbred mice (Levy et al., 1975a; Fischinger et al., 1976). Heatinactivated sera from nineteen Swan mice were tested for. similar activity. The results illustrated in Table 1 show that the sera of all Swan mice, even when only 2 months old, strongly neutralized the xenotropic Ha-SV pseudotype. The neutralization titers varied from 1/320 to more than 1/3200, independently of the ANA titres. Similar titres are regularly found in the sera of C57Bl/6 or C3H/He mice as illustrated in Table 1.

Study of the ecotropic MuLV production in Swan mice Ecotropic MuLV production was individually tested in seventeen ANA-positive Swan mice. In addition, seven Swan mice exceeding 14 months of age were tested because, unusually, these were ANA-

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TABLE 1. Neutralization of xenotropic virus by normal mouse sera

Mouse strain

Age (months) ANA titre Neutralizing titre*

Swan

2

Swan

6

C57B1/6

2

C3H

3

> 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 > 100 > 100 > 100 > 10 < 10 < 10 < 10 < 10 < 10 14 months).

It is unlikely, therefore, that X-MuLV causes autoimmune disorders in Swan mice, since unlike NZB, these mice do not produce high levels of this agent. Indeed Swan mice behave like most inbred strains in this respect. The experiments of Datta & Schwartz (1976) also show a lack ofcorrelation between XMuLV expression and the appearance of autoimmunity in the crosses between NZB and C57B1/6, SWR or B1R.A mice, supporting the idea that X-MuLV do not cause autoimmune diseases in these strains. This may also be true of NZB and (NZB x NZW)F1 mice. Other genetic factors are involved in these strains and in Swan mice. The possible role of ecotropic type C viruses is still more difficult to assess. It is known that the inoculation of ecotropic MuLV increases the ANA titre in various inbred strains of mice (Cannat & Varet, 1972). However, the autoimmune NZB mice apparently does not produce any ecotropic agent (Levy et al., 1975b), whereas those strains which produce high titres, such as AKR and C58 mice, do not show autoimmune features. NZW mice, however, produce high levels of N-tropic MuLV (Stephenson et al., 1975). One could therefore suppose that this agent would be at least responsible for the very high levels of anti-DNA antibodies found in (NZB x NZW)F1 hybrids. Our results do not allow a definite conclusion, since they show on the one hand that Swan mice are certainly only low producers of ecotropic agents, but on the other hand that MuLV production appears to be more frequent in ANA-positive than in ANA-negative mice. Opposite conclusions could be derived from these observations. However, three arguments seem to favour the idea that there is no direct relationship between ecotropic virus expression and the appearance of ANA. Firstly, Swan mice apparently produce no more ecotropic virus than most inbred strains of mice. Secondly, the youngest ANA-positive mice tested, despite a high ANA titre, did not produce ecotropic viruses, showing that the two phenomena are dissociated. Thirdly, considering the very low frequency of producer cells in the spleens, the differences observed between ANA-positive and ANA-negative mice might not be significant. On the whole the role of an ecotropic virus cannot be definitely ruled out from these experiments, but it appears unlikely. Furthermore, it cannot be excluded that the chronic stimulation of lymphoid cells in autoimmune mice would favour the expression of endogenous ecotropic agents, as also observed in other systems (Hirsch, 1973).

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In conclusion, Swan mice are a model of autoimmune animals, with a disease resembling that of (NZB x NZW)F1 hybrids, but without xenotropic type C virus expression and with a production of ecotropic endogeneous type C agents similar to that of the non-autoimmune inbred strains of mice. We thank Ms F. Pozo for expert technical assistance. The work was supported by grants from INSERM and DGRST.

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