key: cord-0998198-s0bxdbzv
authors: Lavi, Ehud; Suzumura, Akio; Murray, Edward M.; Silberberg, Donald H.; Weiss, Susan R.
title: Induciton of MHC class I antigens on glial cells is dependent on persistent mouse hepatitis virus infection
date: 1989-04-30
journal: Journal of Neuroimmunology
DOI: 10.1016/0165-5728(89)90040-4
sha: c8bb18d5a7c69d51bd204887ffa937dbefa2d136
doc_id: 998198
cord_uid: s0bxdbzv

Abstract H-2 class I antigens, but not class II antigens, were detected on the surface of glial cells persistently infected with mouse hepatitis virus strain A59 (MHV-A59) as late as 90 days post-infection. Uninfected glial cells remained negative for H-2 class I and class II surface antigens. We have previously shown that conditioned media from infected glial cell cultures (supernatants) contain a factor unrelated to infectious virus and capable of inducing H-2 class I antigens on uninfected glial cells. The synthesis of this factor appears to be dependent on production of infectious virus since the H-2 inducing activity could not be detected 3 days following the addition of neutralizing antibodies to the cultures. This suggests that H-2 inducing activity contains as unstable component, the synthesis of which is dependent on continual virus production. Persistent MHV infection and H-2 class I antigen expression may play a role in MHV-induced demyelination.

. The chronic disease is characterized by persistence of viral nucleic acid in the white matter (Lavi et al., 1984) . The mechanism of demyelination is unknown. However, in the case of JHM, a closely related strain of MHV, there is evidence both for cytolytic destruction of oligodendrocytes during MHV-induced disease (Lampert et al., 1973) and for a mechanism involving an autoimmune reaction against myelin basic protein (Watanabe et al., 1983) .

MHV infection of glial cells, either cells infected in vitro or cells cultured from the brains of pre-infected mice, results in persistent production of moderate levels of virus with little cytopathic 0165-5728/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division) effect for as long as the cells survive (Lavi et al., 1987) . The infected glial cells, both astrocytes and oligodendrocytes, show detectable levels of H-2 class I, but not class II, surface antigens when assayed either by immunofluorescence or by radioimmunoassay (Suzumura et al., 1986 (Suzumura et al., , 1988 3 days post-infection. In contrast, uninfected glial cells do not contain detectable levels of these antigens. Previously we have shown that H-2 induction was due to a soluble factor(s) synthesized in the infected glial cultures, probably by astrocytes (Suzumura et al., 1986 (Suzumura et al., , 1988 . The activity of the factor was not dependent on infectious virus since infected glial cell supernatants, treated with ultraviolet (UV) light, which inactivates infectious virus, could induce H-2 class I surface antigens when added to uninfected glial cells. This factor was also shown not to be interferon, another factor known to induce H-2, class I antigens on glial cells (Wong et al., 1984; Suzumura et al., 1988) , or virus particles (Suzumura et al., 1988) . In this communication we demonstrate that the continued production of the H-2 inducing factor by cultured glial cells depends on persistent MHV infection.

We have assayed for H-2 class I expression in glial cells at much later times after infection than previously examined. For the experiment described in Fig. 1 , 5-day-old C57BL/6 mice were infected intracerebrally with 400 TCIDs0 of MHV-A59; one day later mixed ghal cultures were derived from these mice by previously described techniques (McCarthy et al., 1980; Suzumura et al., 1984 Suzumura et al., , 1988 . These cultures typically contain > 90% astrocytes as determined by immunostaining with antiserum directed against glial fibrillary acidic protein (GFAP), an astrocyte-specific protein; the remainder of the cells are a mixture of oligodendrocytes, fibroblasts and microglia (Suzumura et al., 1984 (Suzumura et al., , 1985 Lavi et al., 1987) . These cells remained in culture and continued to produce infectious virus for up to 90 days (Fig. 1) . A radioimmunoassay using a monoclonal antibody directed against H-2DbK b (supematant of cell line 28-8-16S, Litton Bionetics) and a secondary 125I-labeled rabbit anti-mouse immunoglobulin (Amersham) were used to measure H-2 class I expression in these cultures 7, 28 and 90 days post-infection (Suzumura and Silberberg, Each column represents mean 125I cpm value of triplicate coverslips after subtraction of parallel mean value of triplicate control coverslips incubated with supernatant from non-producing myeloma SP2/0 cells supplemented with 100/~g/ml of IgG. Supernatants from infected cells at each time point were assayed for infectious virus titer by a TCIDs0 assay in L-2 fibroblasts (Lavi et al., 1987) . For values shown in columns (a), P < 0.001 as compared to controls.

. For up to 90 days after infection (89 days post-culturing), significant levels of H-2 b surface antigens could be detected in the cultures. Expression of H-2, class II surface antigens (as measured by use of a monoclonal antibody directed at Iab (superuatant of cell line 26-18-8S, Litton Bionetics) remained negative throughout the experiment ( Fig. 1, b lanes) as did the expression of H-2, class I ( Fig. 1 ) and class II (data not shown) on uninfected cells. Controls for haplotype specificity were provided by a monoclonal antibody directed against H-2KkD k (supernatant of cell line 16-1-2N, Litton Bionetics). (Positive control for the Iab antibody was provided by reaction with microglia derived from mouse brain. Positive control for the H-2DkK k antibody was provided by reaction with glial cells derived from AKR mice (Suzumura et al., 1985) .) The same results were obtained for cultures that were derived from uninfected mice and subsequently infected in vitro (data not

Expression of H-2 class I in these cultures was detectable on approximately 80% of the cells while only about 20% were viral-antigen positive (as detected by immunofluorescence) as previously described for cultures examined at early times after infection (Suzumura et al., 1986; Lavi et al., 1987) . The majority of H-2 class I-positive cells were astrocytes (as measured by staining with anti-GFAP) as previously described for cultures examined at early times after infection (Suzumura et al., 1985) although some were microglia and oligodendrocytes (not shown). The fact that the majority of cells were positive for H-2 surface antigens, while only 20% expressed viral antigen has been attributed to a soluble factor released by infected cells (Suzumura et al., 1985) .

To determine whether the production of the glial cell factor that induces H-2 expression in these cultures is dependent on continual viral replication, cultures were treated with neutralizing anti-viral antibody (Table 1) . Mixed glial cultures were infected with MHV-A59 (1 TCIDs0/cell ) and incubated at 37°C for 3 days. On days 3 and 6 post-infection, the medium was replaced with fresh medium containing 10% normal rabbit serum or 10% neutralizing anti-MHV-A59 rabbit serum. On day 9 post-infection, the antiserum containing medium was replaced with fresh medium lacking antibodies. The level of infectious virus remained below the level of detection for as long as the antibodies were present in the medium. As shown in Table 1 , during the period of decreased viral production, the glial cell supernatants lacked the ability to induce H-2 antigens when added to uninfected glial cultures as assessed by indirect immunofluorescence. However, by 21 days postinfection, 12 days after the antibody containing media was replaced with fresh media, infectious virus was again produced and culture supernatants regained the ability to induce H-2 when added to uninfected glial cells (Table 1) . Table 1 suggests that it takes longer for detection of the 109 TCIDs0/cell ). The medium was replaced with medium containing 10% normal rabbit serum at PID 3 and again at PID 6. At PID 9, medium containing antiserum was replaced with normal medium.

c Mixed ghal cells were infected with MHV-A69 (MOI = 1 TCIDs0/cell ) and incubated with media containing 10% anti-MHV-A59 serum (neutralization titer of 1:10240) at day 3 and again at day 6. At day 9 medium containing antiserum was replaced with normal medium. This part of the experiment was carried out in duplicate. a PID = post-infection days. e Virus titer contained in supernatants, expressed as TCIDs0/ml, was measured in L-2 mouse fibroblasts (Lavi et al., 1987) .

against H-2DbK b (dilution of 1 : 2) (Suzumura et al., 1986) . Each experimental point represents the average of duplicate coverslips. As negative controls, coverslips were incubated with SP2/0 cell supernatant supplemented with 100 #g/ml IgG. As positive controls for H-2 class I antigen induction coverslips were incubated with 100 #g/ml y-interferon (Suzumura and Silberberg, 1985; Suzumura et al., 1986, H-2 inducing factor after removal of antibody. This could be because it may take time to completely deplete the system of antibody and for viral release to return to previous levels. In the data in Table 1 it is apparent that H-2 inducing factor is detectable either before or at the same time that the titer of viral released is 1.55 × 103. Titers in excess of this may be observed by one day after initial infection. It may be the titer of virus released that is important, not the time period between infection and assay.

We have shown previously (Suzumura et al., 1986) and in Table 1 , that UV-irradiated infected cell supernatant can induce H-2 class I antigens on uninfected glial cells. Two lines of evidence suggest that this induction is not mediated by virus. First, virus particles can be removed from infected-cell supernatants by centrifugation (Suzumura et al., 1988) without affecting the induction of H-2 antigens. Second, treatment of supernatants from infected cells with neutralizing antiviral antiserum does not alter the ability of those supernatants to induce H-2 antigens on uninfected cells (Suzumura et al., 1988) . These observations suggest that the factor in infected-cell supernatants responsible for the induction does not require infectious virus. However, the addition of neutralizing anti-viral antiserum to infected glial cells does prevent the production of a supernatant capable of inducing H-2 antigens on infected cells, suggesting that the synthesis of an active H-2 inducing factor is dependent on the concomitant production of infectious virus. It is possible that the soluble H-2 inducing factor is induced in astrocytes by active viral replication but that the factor is not very stable and therefore continual production of the factor depends on continual viral replication.

These results suggest that persistent coronavirus infection of glial cells results in the continued production of a soluble factor that causes enhanced expression of H-2 class I antigens on glial cells. This expression could result in virus-induced demyelination by allowing cytotoxic T lymphocytes to recognize either a viral or cellular antigen in an H-2-restricted manner and promote the destruction of glial cells. This hypothesis is presently being investigated.

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This study was supported by grants NS 21954, AI 17418 and NS 10037 from the National Institutes of Health. We thank Drs. Philip Zoltick and Jim Gombold for many helpful suggestion and we thank Sonia Estape for preparation of the manuscript.