Lymphocytic choriomeningitis virus-induced alterations of T helper-mediated responses in mice developing autoimmune hemolytic anemia during the course of infection.

The effect of LCMV on CD4+ T lymphocytes was analyzed in C3HeB/FeJ mice after infection with the Docile strain of this virus. Our results indicated that LCMV triggers: i) an inhibition of Th2 lymphocyte differentiation induced by concomitant immunization with a nonviral protein antigen; ii) a depression of T helper-dependent antibody responses elicited by such an immunization; and iii) a CD4+ cell-mediated proliferation of spleen cells leading to increased interleukin-4 and interferon-gamma message expression and IgG2a-restricted total immunoglobulin secretion. Taken together, these results indicate that LCMV profoundly affects CD4+ cell-mediated immune responses in infected animals. Such modulations of T-helper functions may explain the preponderance of IgG2a in the antierythrocyte autoimmune response induced by the virus in C3HeB/FeJ mice.

Infection of C3HeB/FeJ mice with the docile strain of lymphocytic choriomeningitis virus induces autoantibodies specific for erythrocyte Band 3.

C3HeB/FeJ mice infected with the docile strain of lymphocytic choriomeningitis virus (LCMV-d) develop a persistent infection with a transient haemolytic anaemia. Immunoglobulin can be eluted from the red blood cells (RBC) of these mice but it cannot be detected on the RBC by a conventional antiglobulin test. The present study demonstrates that RBC from such mice bear erythrocyte autoantibodies which are predominantly of the IgG2a subclass, with lower levels of autoantibodies of the IgG1, IgG2b and IgG3 subclasses. To identify the target antigen the autoantibodies were eluted from the RBC of LCMV-infected mice. The eluted autoantibody bound to intact normal RBC and precipitated a 105000 MW component that corresponds to murine Band 3 protein. A monoclonal antibody derived from mice infected with LCMV-d also precipitated mouse Band 3, and reacted specifically by enzyme-linked immunosorbent assay against a purified preparation of Band 3. This study has shown that in C3H mice infected with LCMV-d which develop autoimmune haemolytic anaemia, the target autoantigen is erythrocyte membrane Band 3.

Involvement of CD4+ cells in lymphocytic choriomeningitis virus-induced autoimmune anaemia and hypergammaglobulinaemia.

Development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of Lymphocytic Choriomeningitis (LCM) virus. The C3HeB/FeJ and B10. Br/SgSnJ mouse strains have been of special interest because they display autoimmune haemolytic anaemia with varying degrees of apparent immunological involvement. In this report, we examined the role of CD4+ T helper cells in this autoimmune response by treating mice with the CD4-specific GK1.5 monoclonal antibody. We also determined if polyclonal activation of B lymphocytes, induced either by LCM virus or by lactate dehydrogenase-elevating virus, another well known B cell activator, correlated with the development of anaemia in these mice. Our results strengthened the central role of the immune system in the anaemia in C3H mice by showing that depletion of CD4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. As reported by others, we found that the anaemia was more mild in B10.BR mice than in C3H mice. However, we could not confirm the difference in the degree of B lymphocyte polyclonal activation between these mice. Furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma IgG levels.

Influence of viral infection on anti-erythrocyte autoantibody response after immunization of mice with rat red blood cells.

Natural or deliberate activation of the immune system of pathogen-free mice markedly affected their response to an autoimmune-inducing stimulus. Specifically, mice immunized with rat red blood cells were found to make antibodies reactive with both rat and mouse erythrocytes. Animals housed for an extended period in a conventional environment developed an autoimmune response twice as fast as those kept in isolators. In an attempt to emulate this effect, mice kept in a sterile environment were infected with a potent polyclonal activator of B lymphocytes, lactate dehydrogenase-elevating virus, at the same time as they were inoculated with rat erythrocytes. Whereas uninfected animals developed a progressively increasing autoantibody titer, infected mice quickly attained high anti-erythrocyte autoantibody titers that remained rather constant. Contrary to circulating autoantibodies, bound anti-erythrocyte antibodies decreased with time. Virus infection enhanced all the IgG subclass responses, with the exception of IgG1, to both rat and mouse erythrocytes. None of the modifications of the autoimmune responses resulted in anemia.

The presence of an anti-erythrocyte autoantibody in C3HeB/FeJ mice after lymphocytic choriomeningitis virus infection.

Lymphocytic Choriomeningitis (LCM) virus, substrain Docile, causes a chronic infection in adult C3HeB/FeJ mice. The virus also induces a severe anemia which, unlike the viremia, eventually resolves. Initially, there is frank bone marrow deficit, but the anemia persists well beyond a strong erythroid compensatory response. An immune-mediated basis for the hemolytic anemia was suggested by its abrogation in cyclophosphamide-treated mice, as well as an abnormal number of spherocytes in the circulation. We now show by ELISA assay, using either anti-mouse Ig or RBC membrane ghosts as catching antigen, that unusually high quantities of antibodies can be eluted from the RBCs of virus-infected mice. Furthermore, the high transient antibody concentration correlates with the severity of the anemia. With no evidence for complement playing a role in the anemia, these data indicate that erythrophagocytosis (via macrophage FcRs) may be the mechanism for RBC elimination. The possibility of molecular mimicry (antibody cross-reactivity between LCM and RBC membrane epitopes) was considered but appeared unlikely since the RBC antibody eluates gave no signal in an LCM-specific ELISA (which showed an ever increasing serum titer of virus-specific antibody). Isotype determination of the RBC eluates revealed the following: IgG2a much greater than IgG1 greater than IgG2b greater than IgG3 greater than IgM. The precise role, if any, of LCM-virus induced polyclonal activation (most strikingly in the IgG2a subclass) has yet to be determined.

The combination of major histocompatibility complex (MHC) and non-MHC genes influences murine lymphocytic choriomeningitis virus pathogenesis.

Resistance to the acute lethal disease caused by the docile strain of lymphocytic choriomeningitis (LCM) virus varies widely between different mouse strains. In order to study the inheritance of host influence on susceptibility to this strain of LCM virus, we crossed the F1 to the parent with the recessive disease phenotype. In all cases, susceptibility was dominant. In backcross progeny obtained from matings of parental strains differing in both major histocompatibility complex (MHC) and non-MHC (SWR; C3H), 90% of the challenged mice died, indicating that at least three loci controlled susceptibility to the disease. When the parental strains carried similar MHC haplotypes but dissimilar background genes (B10.BR; CBA), 78% of the backcross mice succumbed, indicating that at least two non-MHC loci influenced disease susceptibility. It is unlikely, however, that the same two non-MHC loci are critical in all genetic combinations, since F1 produced from two H-2 identical, resistant strains (B10.BR; C3H) were found to be fully susceptible. When congenic mice, differing only in the D-end of the MHC region, were analysed, 50% of the backcross animals died, indicating that one gene in the MHC region was important; segregation analysis comparing MHC serotype and disease outcome indicated the H-2D locus itself as the determining factor.

Meningeal macrophages reflect lymphocytic choriomeningitis virus pathogenic phenotypes.

Intracerebral (i.c.) infection of adult mice with lymphocytic choriomeningitis (LCM) virus can result in acute lethal central nervous system (CNS) disease which is the result of the host's thymus-derived lymphocyte (T cell) response against the virus. Whether the specific effector function of the T cell is that of a cytotoxic cell (Tc) or a delayed-type hypersensitivity cell (Td) is still under debate. We assumed that if Td cells were important in pathogenesis then accessory cells in the brain (specifically, glass-adherent macrophages) might vary with the outcome of i.c. infection. We found that accumulation of macrophages in the brain (washed from meninges and skull cap) appeared to be independent of the severity of the infection (controlled by the mouse strain as well as the strain and dose of virus used). However, differentiation of macrophages was clearly linked to whether or not the infection caused rapid death. In mice that were destined to survive, macrophages became large, extensively vacuolated, and phagocytically active. In lethally-infected mice macrophages were small and had poor phagocytic abilities. At present this dichotomy could be viewed as either a cause or a consequence of disease outcome. However, the data are not inconsistent with the hypothesis that Td lymphocytes may be of primary importance in pathogenesis.

Lack of correlation between serum titres of interferon alpha, beta, natural killer cell activity and clinical susceptibility in mice infected with two isolates of lymphocytic choriomeningitis virus.

Intracerebral infection of adult immunocompetent mice with most strains of lymphocytic choriomeningitis virus (LCMV) caused a systemic infection and led to severe meningoencephalitis and death due to the induced T cell immune response. The susceptibility of congenic mice to the two plaque variants Docile and Aggressive of LCMV strain UBC was shown to be mouse strain-dependent. To investigate the possible correlation between acid-stable interferon (IFN) and natural killer (NK) cell responses and the susceptibility to the two UBC LCMV substrains, serum titres of acid-stable antiviral activity, presumably IFN-alpha, beta and NK cell activities were determined in various mouse strains at different times after intracerebral infection. The two viral isolates induced comparable IFN-alpha, beta serum titres and caused similar NK activities in the same mouse strain. Between different mouse strains, marked differences in the kinetics and amount of IFN production were observed, yet there was no correlation with the susceptibility to the two UBC LCMV substrains. Additionally, there was no correlation between the magnitude of the IFN-alpha, beta serum titres and the NK activities induced in the spleen by the viral inocula. Overall, the findings suggest that levels of circulating IFN-alpha, beta are only of minor importance for the development of LCM disease.

Different Tc response profiles are associated with survival in the murine lymphocytic choriomeningitis virus infection.

The pathogenicity of lymphocytic choriomeningitis virus (LCMV) inoculated intracerebrally (i.c.) varies with virus strain and dose as well as with the mouse strain used as host. Recently, results have indicated that susceptibility to lethal disease correlates directly the ability of the host to produce early and high virus-specific Tc activity. However, in the present studies we demonstrate that even though this holds true in many mouse/virus combinations, it does not apply in others. Thus, in C3H mice infected with (moderately) high doses of Traub strain LCMV, early and high Tc activity was found despite a mortality rate of only 10-20%. Similarly, in C3H mice inoculated with the aggressive and docile substrains of UBC strain LCMV, which differ markedly in their pathogenicity for this mouse strain, similar kinetics of Tc induction were observed. Finally, in DBA/2 mice which do not die following infection with the otherwise lethal aggressive substrain, Tc induction could be found to be as efficient as in BALB/c mice, all of which die from acute LCM disease when infected with this virus isolate. The results indicate, therefore, that early and high Tc activity does not constitute a sufficient prerequisite for lethal disease, and that different Tc response profiles may be associated with low mortality following i.c. inoculation with LCMV.

Severe and transient pancytopenia associated with a chronic arenavirus infection.

A so-called 'docile' strain of Lymphocytic Choriomeningitis Virus (LCMV) lacks the ability to cause the fatal central nervous system syndrome, commonly associated with most other strains of this virus, in C3HeB/FeJ mice. Hematological evaluation during a 5 week period revealed that every mouse experienced a pancytopenia which was the most severe around three weeks post-infection. The abnormal red blood cell (RBC) morphology seen in the peripheral blood along with the increased reticulocyte count and marked erythroid hyperplasia in the bone marrow indicated peripheral destruction, rather than stem cell inhibition, as the mechanism causing the anemia. An increase in the 59Fe uptake into the blood confirmed the fact that there was no loss in the erythropoietic capabilities in these mice at this time. Although it was clear that the RBCs were being destroyed in the periphery, there was no evidence of a microangiopathic hemolytic anemia nor of a direct viral infection of these cells. Cyclophosphamide treatment, however, prevented the phenomenon. Thus, it seemed likely that the virus-induced hemolytic anemia in these mice was immune-mediated. The late, but not the early drop in the white blood cell counts and the thrombocytopenia, on the other hand, could be traced to granulocyte and megakaryocyte inhibition in the bone marrow.

Susceptibility to murine lymphocytic choriomeningitis maps to class I MHC genes--a model for MHC/disease associations.

Susceptibility to some human diseases is linked, albeit weakly, to major transplantation antigens (HLA) encoded by the major histocompatibility gene complex (MHC). Here we have studied MHC/disease association in inbred strains of mice after intracerebral (i.c.) injection of lymphocytic choriomeningitis virus (LCMV). This route of infection leads to a lymphocytic choriomeningitis (LCM) which is not the result of direct cytopathic effects of the virus but is caused by the induced T-cell immune response: immunocompetent mice die whereas T-cell-deficient mice survive. By using two plaque variants of LCMV strain UBC (refs 7,8), we found that susceptibility to LCM was dependent on the LCMV strain used ('aggressive' versus 'docile' UBC-LCMV) and on the various genes of the host mouse strains. In addition, susceptibility to LCM caused by docile UBC-LCMV was clearly linked to the murine major histocompatibility locus H-2D: in MHC-congeneic C57BL/10 mice, susceptibility correlated with early onset and high activity of measurable LCMV-specific cytotoxic T cells in meninges and spleens and could be mapped to H-2D. This model shows that a severe immunopathologically mediated clinical disease in mice can be regulated directly by MHC genes of class I type and supports the notion that many MHC/disease associations directly reflect MHC-restricted and MHC-regulated T-cell reactivity.

Lack of correlation between cytotoxic T lymphocytes and lethal murine lymphocytic choriomeningitis.

Adoptive transfer of lymph node and spleen cells from mice infected with LCM virus to similarly infected immunocompromised recipients has been the classic way to demonstrate the lethal role of T cells in the CNS disease caused by this virus. Isolation and adoptive transfer techniques are presented here which show that Thy-1+ cells isolated from the meningeal infiltrates (MI) of LCM virus-infected mice possess this property. We compared various T cell functions of MI cells taken from mice infected with two strains of LCM virus differing markedly in their pathogenicities. One of these strains, termed aggressive, caused a typical, invariably fatal, CNS disease within 7 to 10 days after infection. The other virus, termed docile, killed few mice after the standard intracerebral inoculation, and could persist in the mice for 6 mo or more. The yields of MI leukocytes from mice infected with docile virus varied from 50 to 100% of those found in mice infected with aggressive virus (3 X 10(6) cells/brain). On a cell-to-cell basis, the CTL activity in the MI of mice infected with docile virus ranged from 50 to 100% of that found in the MI of mice infected with aggressive virus. MI cells from mice infected with aggressive virus consistently caused lethal disease by adoptive transfer into immunocompromised (irradiated) recipients infected with either strain of virus. All attempts to induce lethal disease by adoptive transfer of MI cells (or splenocytes) from mice infected with docile virus into irradiated recipients failed. The latter experiments with the docile-MI cells were performed with six times the number of aggressive-MI cells needed to kill irradiated recipients by adoptive transfer. The possible reasons for this discordance between CTL and in vivo killer function are discussed.

Immune recognition of tumor cells in mice infected with Pichinde virus.

Pichinde virus (PV), a member of the Arenaviridae family, protects mice from a lethal inoculation with the sarcoma 180 (S180) tumor cell line. Virus replication, which is required for protection, occurs primarily in the spleen and tumor. During the first 4 days, elevated natural killer (NK) cell activity parallels an increase in serum interferon in PV-infected mice. On day 7 after infection virus-specific cytotoxic T cells (CTLs) are found in the mouse. This strong response peaks on day 13 and gradually declines over the next 17 days. The tumor-specific CTL response appears more slowly and is less intense than the virus-specific response, especially in the uninfected mouse. However, CTLs from either type of mouse recognize PV-infected tissue culture S180 target cells better than uninfected ones. Even though the primary tumor-specific immune response appears weak, mice that have cleared both virus and tumor are refractory to a subsequent challenge with S180 cells and rapidly produce tumor-specific CTLs. Thus, our data indicate a number of ways in which virus infection could lead to immune elimination of tumors: (1) Virus-induced interferon stimulates NK-cell activity, which in turn could control tumor load until a specific response is mounted against the S180 cells; (2) early onset of the tumor-specific T-cell response could be brought about by viral-enhanced tumor antigen presentation to the immune system; and (3) the tumor-specific T-cell response could be augmented through a "bystander' phenomenon involving factors associated with T cells responding specifically and vigorously to the virus itself.

Lethal role of interferon in lymphocytic choriomeningitis virus-induced encephalitis.

After intracerebral inoculation of adult C3H mice, the 'docile' strain of lymphocytic choriomeningitis (LCM) virus multiplied to high titre in several visceral organs. Although the virus content of lung, liver, spleen and brain was high, these mice did not die but became long-term carriers of the virus. Injection of mice with the same dose of the 'aggressive' strain of LCM virus resulted in much lower virus titres in these organs; nevertheless, 100% of the mice died within 7 to 9 days. The results presented here show that mice infected with the 'aggressive' virus do not die if treated with anti-interferon globulin. Under these conditions the titres of 'aggressive' virus were as high in the different organs as in mice injected with the 'docile' virus. These results are consistent with the hypothesis that inhibition of LCM virus multiplication in various organs by interferon results in a lethal disease. The possible mechanisms underlying this seemingly paradoxical phenomenon are discussed.

Chronic retinitis in rats infected as neonates with lymphocytic choriomeningitis virus: a clinical, histopathologic, and electroretinographic study.

The long-term sequelae to infection of neonatal rats with lymphocytic choriomeningitis virus were studied by a variety of approaches, including indirect ophthalmoscopic, electroretinographic, and histopathologic methods. Data from these studies demonstrated that a progressive chronic retinitis develops after the acute, virus-specific, immune-mediated retinopathy. This chronic inflammation eventually leads to a total destruction of the retinal architecture. An autoimmune reaction against normally sequestered retinal antigens, released during the acute state of necrotizing retinitis, is probably the initiating mechanism of the chronic disease. This experimental disease, triggered by infection with a relatively harmless virus, constitutes a very convenient animal model of chronic retinitis.

Lymphocytic choriomeningitis virus killer T cells are lethal only in weakly disseminated murine infections.

Two types of lymphocytic choriomeningitis (LCM) viruses were studied which, upon intracerebral injection into adult C3H mice, provoked either (a) acute fatal central nervous system (CNS) disease or (b) life-long persistent infection. Both virus types, (a) aggressive and (b) docile, had been found to induce LCM-specific lymphocytes with comparable in vitro lytic activity (11). Because the requirement for T cells in the development of adult LCM disease has been extensively documented, we sought other reasons for the lack of acute disease in mice infected with docile virus. A striking correlation was found between the outcome of the infection and spread of virus to visceral organs. Adoptive transfer experiments showed that a 300-plaque forming unit inoculum of docile virus induced a population of T cells in donor mice fully capable of causing CNS disease in identically infected recipients. This disease causing ability was lost if the interaction was delayed beyond 3 d after infection of the recipients, but could be preserved by lowering the size of the viral inoculum in the recipients. Furthermore, without adoptive transfer, very low intracerebral doses of docile virus (which mimicked the normally slow spread of aggressive virus) were lethal. On the other hand, very high doses of aggressive virus, which mimicked the normally rapid spread of docile virus, did not induce fatal CNS disease. The results suggest that rapid dissemination of the LCM infection creates multiple target organs which divert the focused lethal T cell attack on the brain.

Cytotoxic T cells are induced in mice infected with lymphocytic choriomeningitis virus strains of markedly different pathogenicities.

The ability of two lymphocytic choriomeningitis virus substrains to induce cytotoxic T-lymphocyte (CTL) responses in intracerebrally infected mice was examined. One strain, designated A (aggressive), provoked a convulsive type of death in 100% of the mice within 8 to 9 days, whereas the other strain, designated D (docile), killed less than 10% of the mice during 28-day observation periods. CTL activity was assessed by the capacity of partially purified splenocytes to lyse 51Cr-labeled L-cell targets infected with either type of lymphocytic choriomeningitis substrain. The CTL population was identified by its sensitivity to anti-Thy-1 serum and its inability to lyse uninfected target cells or infected target cells with which it differed at the level of antigens controlled by the major histocompatibility gene complex. A strong CTL response developed in mice infected with either lymphocytic choriomeningitis substrain, although the activity provoked by substrain D was somewhat less than that seen after substrain A infection. Peak CTL activities induced by both strains occurred at about the same time. Even though docile virus replicated more extensively in the brain than did aggressive virus and fluorescent antibody staining revealed similar distributions of viral antigen, no inflammatory response was noted in the brains of mice infected with docile virus. These results are discussed with regard to the role of CTLs in mediating classic central nervous system pathology.

Interferon induction by lymphocytic choriomeningitis viruses correlates with maximum virulence.

Lymphocytic choriomeningitis (LCM) viruses isolated from the blood of persistently infected mice, could be clearly divided into two categories by observing the disease patterns they produced after intracerebral (i.c.) injection in a number of adult inbred and outbred mice. One type (aggressive) caused the classic pattern of convulsive death in 100% of the mice 7 to 9 days after infection, while the other (docile) caused a protracted disease with deaths occurring, if at all, 2 to 4 weeks after infection. Interferon could be detected in the serum of adult mice on the 3rd and 4th day after infection with several independently cloned aggressive, but not docile, viruses. The inability of docile virus to induce interferon was not due to poor or delayed virus replication in the brain. The aggressive pattern of disease could be provoked easily in docile virus-infected mice with the interferon inducers poly(rI) . poly(rC), tilorone hydrochloride or Newcastle disease virus. The amount of interferon produced had little effect on the mean day of death. Mice that differed over 10-fold in their serum interferon levels after LCM infection, either by genetic predisposition or by stimulation with increasing amounts of poly(rI) . poly(rC), presented almost identical patterns of mortality.