key: cord-0763712-avdp8gnt authors: nan title: Inducibility of Ia antigen on astrocytes by murine coronavirus JHM is rat strain dependent date: 1987-07-01 journal: J Exp Med DOI: nan sha: ec0a48de352326aecc319e76ea1f47b989ffcd8f doc_id: 763712 cord_uid: avdp8gnt Inducibility of Ia molecules on cultivated astrocytes by JHM virus correlates with demyelinating disease susceptibility of animals from which these astrocytes are derived. On the contrary, class I induction of both astrocytes and oligodendrocytes occurs as a consequence of normal cultivation procedures in both susceptible and resistant strains. Increased expression of class I antigens on rat astrocytes and oligodendrocytes is not related to JHM viral infection as it is in the mouse. These data indicate that strain differences in Ia inducibility, rather than inducibility of class I antigens, by JHM virus may explain higher levels of T cell-mediated damage to myelin during infection in susceptible rat strains compared with resistant strains. Infection of Lewis rats with the murine coronavirus JHM leads to a persistent infection in the central nervous system (CNS) and a disease process that shares some features with experimental allergic encephalomyelitis (EAE) (1) (2) (3) (4) . After an incubation period of weeks, a subacute demyelinating encephalomyelitis (SDE) develops that is characterized by paralysis (2) . Histologically, the main lesion consists of a primary demyelination associated with perivascular cuffings and a cellular infiltration of macrophages as well as helper and cytotoxic T lymphocytes (1, 2) . SDE animals reveal a cell-mediated immune (CMI) response to myelin basic protein (MBP) as documented in adoptive transfer experiments (1) . In addition, genetic experiments demonstrate that Brown-Norway (BN) rats are resistant to SDE, similar to what has been observed in EAE (4) . These findings indicate an immunopathological reaction in coronavirus-induced SDE in Lewis rats that is lacking in BN rats. A CMI response is genetically controlled at at least two levels : (a) the ability of the animal to generate an encephalolytogenic T cell response to antigen within the brain (foreign or autoantigen) from its genetically determined T cell repertoire and (b) the degree to which the antigen is presented to these T cells in the context of class I and II (Ia) histocompatibility molecules within a particular tissue . Class II-restricted T cells initiate an immune response to infection and both class I-and II-restricted T cells provide cytolysis of infected cells (5) . Since la molecules are normally lacking on cells of the brain (6), the induction of la within the brain is crucial for the initial local presentation of viral antigen during infection . It may therefore be important that astrocytes of Lewis rat brain can be induced to express la directly by JHM virus (7, 8) as well as by other viruses. IFN-y subsequently released by antigen-activated T cells can further induce the antigen-presenting function of astrocytes (9) (10) (11) . The direct induction of la on astrocytes by JHM virus is also thought to play a crucial role in eliciting the excessive T cell response in susceptible rats . Supporting this notion are recent observations by us (12) showing that astrocytes of rat and mouse strains susceptible to EAE express much higher levels of la than astrocytes of resistant strains after treatment with IFN-y. We now extend these studies and analyze JHM virus induction of la on astrocytes of rats susceptible or resistant to SDE and show that, unlike astrocytes of susceptible Lewis rats, induction of astrocyte la by JHM virus is not detectable in the resistant BN rat. Animals . Lewis, BN, and congenic Lewis .BN rats were obtained from the Central Institute for Experimental Animal Breeding, Hannover, Federal Republic of Germany . Tissue Cultures. Mixed glial cultures containing astrocytes and oligodendrocytes were prepared from brainstem and cerebella of 1-d-old rat pups (specific pathogen-free) as recently described (8) . Cultures were incubated at 37°C with 10% C0 2 at maximum humidity, then fed with fresh medium 4 d after plating . Infection of cultures with wild-typeJHM coronavirus began at 5 d after plating. DME with 15% FCS was used as medium . Pure rat astrocyte cultures were prepared from 1 -d-old rat pups as previously described (12) . Pure oligodendrocyte cultures were prepared from whole brains of 4 adult Lewis and BN rats (5-6 weeks old) following the procedure of Kim et al ., 1983 (13) . Virus . Wild type murine coronavirus JHM stock virus was isolated and cloned as previously described (3). Preparation of Conditioned Supernatants. Mixed glial cultures grown for 5 d were infected with wild-type JHM virus at 10 5 PFU/ml, then fed fresh medium . Cultures were incubated thereafter for 5 d when cytopathic effects (CPE) covered 50% of the cell culture area . At this time the virus-containing supernatants were titered and aliquots were stored in liquid nitrogen . As a control, supernatants conditioned over the same 5-d period by uninfected glial cultures were similarly handled. Muramyl Dipeptide Treatment. 7-d mixed glial cultures were treated daily with 0 .1 jug/ml muramyl dipeptide (Sigma Chemical Co ., St . Louis, MO) as recently reported (8), then they were stained for Ia antigens at day 5 after treatment . Fluorescence-activated Flow Cytometry . At 5 d after infection, the glial cultures were prepared for flow cytometry (EPICS V ; Coulter Electronics, Hialeah, FL), as previously described (12) . la and class I molecules were stained using the mAbs Ox6 (14) and Ox 18 (15) , respectively . Pure astrocyte cultures were also stained for Ran-2 antigen (16) and oligodendrocytes for galactocerebroside (17) . Strain-specific Ia Induction ofAstrocytes by JHM Virus . Primary rat glial cultures produced from SDE-susceptible (Lewis and congenic Lewis .BN) and SDE-resistant (BN) rats were treated daily with wild-type JHM virus, and analyzed 5 d after infection . As shown in Fig . 1 , uninfected control Lewis glial cultures typically contained no la' cells ( Fig . I a) , but at 5 d after infection, la' astrocytes became Cell type Oligodendrocyte cultures were >90% positive for galactocerebroside, and astrocytes were >90% positive for Ran-2 antigen stained as described in Materials and Methods . detectable ( Fig. 1 b) , as previously reported (7, 8) . In contrast, in glial cultures derived from disease-resistant BN rats, la was never observed after infection ( Fig. 1 d) with JHM virus. However, infection of cultures of congenic Lewis.BN rats led to astrocyte la induction to the same levels seen in the Lewis rat (Fig. 1 , e and f). Therefore, responsiveness to JHM virus with respect to la induction is controlled by Lewis rat genes located outside the RT-1 locus, and perhaps is related to disease susceptibility of these animals (4). Class I Antigen Expression on Glial Cells. The induction of class I molecules on mouse glial cells by a virus related toJHM virus, coronavirus A59, has recently been reported (18) . In the rat, class I antigens were spontaneously expressed in high constitutive levels on numerous glial cells in control cultures (Fig. 2) , in agreement with others (19) . As shown in Table I , spontaneous induction of class I antigens on pure astrocyte cultures increased over time after plating. Likewise, purified rat oligodendrocytes became strongly class I antigen-positive within 18 h after plating, had increased positivity at 2 d, and were stainable at least 7 d after plating (Table I) . This indicates that pure rat oligodendrocytes rapidly become class I-positive by conditions associated with cultivation and also that soluble class I inducers originating from other cell types were not involved in the rat, as they are in the mouse (18) . With respect to possible enhanced expression of the existing levels of class I antigens as a result of infection (18) , Fig . 2 shows that control glial cellconditioned supernatants containing no JHM virus induced class I antigens on naive cultures (Fig. 2a) to the same degree as virus containing supernatants in Lewis (Fig. 2b) , BN (2c), and congenic Lewis.BN cultures (Fig. 2d) . Therefore, no increase in either the percentage or fluorescence intensity of class I antigenpositive cells could be attributed to JHM virus . The shift of the entire population of fluorescing cells to higher fluorescence intensity channels produced by conditioned supernatant is consistent with the enhancement of class I antigens primarily on spontaneously expressing cells (Fig. 2) . Strain-specific Induction ofIa Molecules by Muramyl Dipeptide. We have recently reported (8) the capacity of the bacterial immune adjuvant, muramyl dipeptide (adjuvant peptide) to induce la molecules on astrocytes in a manner identical to that seen with JHM virus . As presented in Table II , muramyl dipeptide induces astrocyte la in the same strain-specific pattern as does JHM virus . This gives a further indication that the inducibility of la antigens on astrocytes of diseasesusceptible animals compared with resistant animals is not dependent on any one viral or bacterial inducer but related to strain-specific regulation of la molecules expressed at the level of the astrocyte . In this report we have analyzed the effect of JHM virus on la and class I antigen expression on glial cells of different rat strains . We show that JHM virus induces astrocyte la expression in strains susceptible to JHM virus-induced demyelinating disease but not in a resistant strain . Expression of class I antigens was different in the sense that class I genes were expressed before and after infection . Moreover, the expression of class I-positive cells of all rat strains was not appreciably increased by JHM virus above already high levels seen on control glial cells. The similar la-inductive capacity seen between JHM virus and muramyl dipeptide might provide clues to the role ofJHM virus in eliciting autoimmune Medium Demyelination caused by JHM virus infection of Lewis rat brain is mediated by T lymphocytes sensitized to both myelin basic protein and viral antigens (1, 4) . According to our findings, the ability of brain cells to present antigen in the context of class I antigens would appear to be similar between strains, not differing in relation to genetically controlled susceptibility to demyelinating disease. However, the differing degree to which JHM virus induces la antigens on astrocytes in susceptible and resistant strains, as described here, would have a major influence on la-restricted T cell activation and tissue damage . Inducibility of la molecules on cultivated astrocytes by JHM virus (7) correlates with demyelinating disease susceptibility of animals from which these astrocytes are derived. On the contrary, class I induction of both astrocytes and oligodendrocytes occurs as a consequence of normal cultivation procedures in both susceptible and resistant strains. Increased expression of class I antigens on rat astrocytes and oligodendrocytes is not related to JHM viral infection as it is in the mouse (18) . These data indicate that strain differences in la inducibility, rather than inducibility of class I antigens, by JHM virus may explain higher levels of T cell-mediated damage to myelin during infection in susceptible rat strains compared with resistant strains. Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis Relapsing subacute demyelinating encephalomyelitis in rats during the course of coronavirus JHM infection Genetic variation of neurotropic and non-neurotropic murine coronaviruses Coronavirus infection of rats : a model for virus induced demyelinating disease associated with autoimmunity Features of target cell lysis by class I and class 11 MHC-restricted cytolytic T lymphocytes Distribution and quantitation of HLA-A, B, C and DR (la) antigens on human kidney and other tissues . Transplantation (Baltimore) Viral particles induce Ia antigen expression on astrocytes Analysis ofla induction on Lewis rat astrocytes in vitro by virus particles and bacterial adjuvants Astrocytes present myelin basic protein to encephalitogenic T cell lines Expression of Ia antigens by cultured astrocytes treated with gamma-interferon Interferon-gamma induces the expression of H-2 and Ia antigens on brain cells Hyperinducibility of la antigen on astrocytes correlates with strain specific susceptibility to experimental autoimmune encephalomyelitis (EAE) Longter m culture of human oligodendrocytes: Isolation, growth and identification Identificatio n of glycoproteins in rat thymus and purification from rat spleen Mouse monoclonal antibodies against rat major histocompatibility antigens . Two Ia antigens and expression of la and class I antigens in rat thymus Rat neural antigen-2 (Ran-2), a cell surface antigen on astrocytes, ependymal cells, Milller cells and lepto-meninges defined by a monoclonal antibody Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside Coronavirus infection induces H-2 antigen expression on oligodendrocytes and astrocytes Astrocytes as antigen-presenting cells . Part II: Unlike H-2K-dependent cytotoxic T cells, H-2 Ia-restricted T cells are only stimulated in the presence of interferongamma We acknowledge the technical assistance of Ines Tschertner, Erhard Kress for assistance with the EPICS V, and Helga Kriesinger for typing the manuscript .Received for publication 23 February 1987 and in revised form 29 April 1987.