Murine AIDS Research Articles

Contribution of Thy-1−CD4+T Cells to the Disorganization of Lymphoid Follicles in Retrovirus-Induced Immunodeficiency Syndrome, MAIDS

The destruction of the lymphoid follicle (LF) and of the follicular dendritic cell network in the secondary lymphoid organs are major pathological features in murine acquired immunodeficiency syndrome (MAIDS). These pathological changes are associated with functional deficiency of lymphocytes. However, the mechanisms involved have not been established. In MAIDS-susceptible C57BL/6 (B6) mice, the destruction of the LF occurred as early as 2–3 weeks after the infection by LP-BM5 murine leukemia virus (MuLV) and was accompanied by the intrafollicular infiltration of T cells of an unusual phenotype, Thy-1−CD4+. B6 mice, which Thy-1+T cells had been depleted by the repeated inoculation with monoclonal antibody to Thy-1 antigen, developed LF destruction after infection similar to the control B6 mice. The LP-BM5 MuLV-infected C57BL/6-nu/nu(B6-nu/nu) mice, otherwise resistant to MAIDS induction, also developed these abnormalities following the adoptive transfer of highly purified Thy-1−CD4+T cells obtained from MAIDS mice. Thus, the production of Thy-1−CD4+T cells and their infiltration into LF are thought to be involved in triggering the LF destruction. However, the cytolysis of follicular components by activated CD8+T cells or by LP-BM5 MuLV itself was a less likely mechanism as MAIDS developed in B6 mice depleted of CD8+T cell. A/J mice, which are resistant to viral infection in the presence of CD8+T cells, also developed MAIDS and LF destruction after the depletion of CD8+T cells. Furthermore, no follicular destruction was observed in infected B6-nu/numice even though they are highly sensitive to LP-BM5 MuLV.

Th0-like CD4+ T cells protect mice with murine retrovirus-induced immunodeficiency syndrome (MAIDS) against co-infection with Listeria monocytogenes.

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP-BM5 murine leukaemia virus (MuLv) infection. Although LP-BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell-mediated immunity, the mice with established MAIDS infected with LP-BM5 8 weeks previously, showed resistance to an intraperitoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed-type hypersensitivity response to heat-killed Listeria (HKL.) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+ alpha beta T cells but neither by gamma delta T cells nor natural killer (NK) cells. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) were produced by CD4+ T cells from Listeria-infected MAIDS mice in response to the in vitro stimulation with HKL, whereas IFN-gamma but not IL-10 were produced by those from Listeria-infected control mice. These results suggest that T-helper 0 (Th0)-like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice.

Apoptosis by CD95 (Fas)-dependent and -independent mechanisms in Peyer's patch lymphocytes in murine retrovirus-induced immunodeficiency syndrome.

CD95 (Fas)/CD95 ligand (CD95 L)-mediated apoptosis is thought to be involved in the delayed progression of murine AIDS (MAIDS) induced by LP-BM5 murine leukemia virus (MuLV). We show evidence of apoptosis in lymphocytes of Peyer's patches (PP) at the early stage of MAIDS. Both T and B cells in PP expressed CD95 at the early stage of MAIDS and decreased in number thereafter. The decrease in T cells was not evident in CD95-mutated lpr mice with MAIDS, suggesting that CD95/CD95 L interaction is involved in the apoptosis of T cells in PP during the course of MAIDS. On the other hand, the number of B cells was also decreased in PP of lpr mice with MAIDS. The proliferative ability of B cells in PP of MAIDS mice in response to immunoglobulin M cross-linking or lipopolysaccharide was severely impaired, while the B cells normally proliferated in response to anti-CD40 monoclonal antibody. These findings imply that aberrantly activated B cells in PP undergo apoptosis independently of the CD95/CD95 L system during the course of infection with MAIDS virus.

Induction of murine acquired immunodeficiency syndrome (MAIDS) in allophenic mice generated from strains susceptible and resistant to disease.

To examine whether a retroviral disease can be controlled in animals in which cells from a resistant strain coexist in a state of immunological tolerance with cells from a susceptible strain, allophenic mice were constructed and infected with LP-BM5 murine leukemia viruses which induce a fatal disorder, termed murine acquired immunodeficiency syndrome (MAIDS), characterized by lymphoproliferation and immunodeficiency in susceptible inbred strains of mice. We found that in two different strain combinations, resistance to MAIDS was contingent on the presence in individual animals of >50% of lymphocytes of resistant strain origin and correlated with reduction or elimination of retrovirus. In contrast, animals harboring substantial, but less than predominant, numbers of genetically resistant lymphocytes developed disease and died within the same time frame as susceptible control mice with uncontained proliferation of retrovirus.

Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus.

The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.

Therapeutic effects of glycyrrhizin in mice infected with LP-BM5 murine retrovirus and mechanisms involved in the prevention of disease progression.

Glycyrrhizin (GL), a plant extract, has been evaluated for its inhibitory effect on HIV replication in vitro and for its improvement of clinical symptoms in HIV-infected patients. In this study, we used GL in a murine AIDS model (MAIDS) to evaluate these effects. C57BL/6 mice were inoculated with LP-BM5 murine leukemia virus to cause MAIDS. Treatment with GL supplemented with glycine and cysteine (Stronger Neo-Minophagen C, SNMC) was then begun on day 0 or 4 wks after virus inoculation. SNMC was administered three times a week for up to 19 wks. Immunological abnormalities were monitored with respect to the surface phenotype identified by two-color staining for CD3 and IL-2 receptor beta-chain. All mice infected with the virus alone developed MAIDS and died by 14 wks after infection. The immunopathogenesis was estimated to be an abnormal expansion of intermediate CD3 cells (i.e., extrathymic T cells) as well as other types of lymphocytes. SNMC did not change the total mortality rate. However, some mice that began the treatment on day 0 or 4 wks after infection survived 3 wks longer. Splenomegaly and lymphadenopathy in such mice were suppressed. These mice showed normal phenotypic features and normal responses to Con A. These results suggest that SNMC is effective in some MAIDS mice in preventing the progression of disease. When lymphocytes isolated from the liver, spleen and lymph nodes of diseased mice were cultured in vitro, they showed a spontaneous proliferation. Interestingly, such proliferation was inhibited by addition of liver lymphocytes, but not splenic lymphocytes, obtained from normal or SNMC-treated mice. Since liver lymphocytes contains intermediate CD3 cells with autoreactivity, they may possibly suppress the progression of disease.

Endogenous Mtv-encoded superantigens are not required for development of murine AIDS.

Immune activation in murine AIDS (MAIDS) has been suggested to involve a superantigen (SAG). The possibility that SAGs encoded by mammary tumor virus (MTV) might be the source of stimulation was studied by using Mtv mice. Mtv- mice developed typical MAIDS, excluding a requirement for Mtv-encoded SAGs in the pathogenesis of this disorder.

Antiviral activity of biological response modifiers in a murine model of aids. requirement for augmentation of natural killer cell activity and synergy with oral AZT

We employed the Rauscher murine leukemia virus (RMuLV) as a murine retrovirus model of AIDS, to test biological response modifiers (BRM) and antiviral agents for potential therapeutic activity against the human immunodeficiency virus (HIV). We examined the relationship between the augmentation of natural killer (NK) cell activity and antiviral efficacy of a series of BRM, most of which are known inducers of interferon, in this model. Poly [I,C]-LC, MVE-2, and CL 246,738, but not Ampligen, soluble glucan, or 7-thia-8-oxoguanosine, consistently produced antiviral activity. In addition, the combination of suboptimal doses of oral 3'-azido-3'-deoxythymidine (AZT) (in drinking water) and poly [1,C]-LC produced a synergistic antiviral effect. With all the BRM tested, a consistent pattern emerged, namely that antiviral activity always correlated with the augmentation of splenic NK cell activity in infected animals. For instance, poly [I,C]-LC boosted NK activity much more in infected mice treated therapeutically (treatment initiated after infection) than prophylactically (treatment initiated before infection), and it had greater antiviral activity therapeutically than prophylactically. For the BRM tested, antiviral activity did not occur without augmentation of NK activity in infected mice. In contrast, augmentation of NK activity in uninfected mice bore no relationship to antiviral activity. Furthermore, elimination of NK cells by treating mice with anti-asialo GM 1 abolished the antiviral activity of poly [I,C]-LC. Although splenic NK activity was ablated by anti-asialo GM, serum interferon levels were not affected by this treatment. These results point to a causal connection between the augmentation of NK cell activity and the antiviral efficacy of these BRM in this murine AIDS model. NK cells thus appear to play a key role in resistance to this retrovirus, as has been suggested for HIV.

Vitamin E supplementation normalizes immune dysfunction in murine AIDS induced by LP-BM5 retrovirus infection

It is known that murine AIDS, induced by i.p. injection of LP-BM5 retrovirus, is functionally similar to human AIDS. In this study, we tried to examine the effect of vitamin E (dl-α-tocopheryl acetate) supplementation on the decrease of cellular immune functions following the development of murine AIDS. Female C57 BL 6 mice, 4 weeks old, were infected with LP-BM5 retrovirus and then fed control (50 IU/kg diet) or high vitamin E (500 or 2500 IU/kg diet) diets for 10 weeks. The spleen weight and number of splenocytes were largely increased following the development of murine AIDS. On the contrary, vitamin E supplementation suppressed the enlargement of spleen and the increased number of splenocytes following retrovirus infection. The decrease of NK activity shown in mice infected with LP-BM5 retrovirus was also partly improved by high vitamin E diet. Proliferation of splenic T lymphocytes, showing the marked decrease following murine AIDS, was significantly restored by higher vitamin E (2500 IU/kg diet) diet compared to control group, which was still lower in comparison with that of uninfected control group. Furthermore, vitamin E supplementation increased production of interferon-γ (IFN-γ) and suppressed production of tumor necrosis factor-α (TNF-α) from splenocytes. In addition, high vitamin E diet also decreased the increased ratio of CD4 and CD8 single positive T cells following the development of murine AIDS, which was almost equal to the levels of uninfected control and high vitamin E groups. These results suggest that vitamin E supplementation normalizes the decrease of immune functions following the development of murine AIDS.

Inhibition of murine AIDS by reduced glutathione.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome

To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of L-NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFN gamma + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages, from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by L-NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome

To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of l-NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFNγ + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by l-NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.

Mapping of a murine AIDS virus-related proviral gene (Mrv6) in NOD/Lt mice to Chromosome 14

Mapping of a murine AIDS virus-related proviral gene (Mrv6) in NOD/Lt mice to Chromosome 14

Antiviral efficacy and toxicity of ribavirin in murine acquired immunodeficiency syndrome model.

The antiretroviral efficacy and hematotoxicity of ribavirin, a guanosine analogue, have been evaluated in mice infected with the LP-BM5 virus pool [murine acquired immunodeficiency syndrome (MAIDS) model]. Doses ranging from 6.25 to 200 mg/kg/day were injected intraperitoneally twice a day for 6 weeks to infected mice. Drug treatment induced a significant protection against splenomegaly and lymphadenopathy at doses > or = 25 mg/kg. Moreover, doses starting at 50 mg/kg protected against hypergammaglobulinemia, minimized the loss of spleen CD8+ T cells, and reconstituted the capacity of splenocytes to proliferate in response to concanavalin A. The spleen and cervical lymph node architectures were protected, and a reduction in the emergence of germinal centers was observed at 50 mg/kg ribavirin. Hematotoxicity appeared at doses > or = 50 mg/kg ribavirin, and severe anemia was predominant only at doses of 100 and 200 mg/kg. This study shows that ribavirin protects mice against the effects resulting from retrovirus infection at doses of > or = 50 mg/kg in a MAIDS model and induces severe hematotoxicity at doses > or = 100 mg/kg.

TCR triggering of anergic CD4 T cells in murine AIDS induces apoptosis rather than cytokine synthesis and proliferation.

Murine AIDS (MAIDS) induced by infection of C57BL/6 mice with a mixture of retroviruses known as LP-BM5 is characterized by lymphadenopathy, splenomegaly, and T and B cell dysfunction. By labeling with bromodeoxyuridine in vivo, we found vigorous CD4 T cell proliferation during the initial stages of infection, yet a loss in their ability to function both in vivo and in vitro. In addition, a significant fraction of the CD4 T cell population in infected mice undergoes spontaneous apoptosis in vivo. Upon in vitro stimulation with anti-CD3 plus PMA, anergic CD4 T cells from mice with MAIDS fail to progress through the cell cycle (G0/G1 arrest), and a fraction of the cells undergoes apoptosis. The addition of IL-2 along with TCR-mediated stimulation not only fails to rescue CD4 T cells from apoptosis, but enhances activation-induced cell death. To further understand the regulation of the suicide pathway(s) of anergic CD4 T cells vs the cytokine synthesis pathway(s) of normal CD4 T cells, we evaluated their expression of Bcl-2 protein. As infection progresses, the expression of Bcl-2 among CD4 T cells declines and drops further when CD4 T cells are restimulated through the TCR in vitro. These results suggest that this CD4 T cell immunodeficiency in MAIDS includes a TCR-induced program of activation-induced cell death and an uncoupling from cytokine synthesis pathways and proliferation of CD4 T cells. The decline in Bcl-2 expression may be in part responsible for this reprogramming.

Alpha/beta interferons increase host resistance to murine AIDS

Murine AIDS (MAIDS) is caused by a defective retrovirus present in the LP-BM5 murine leukemia virus mixture. Strains of inbred mice differ in resistance to MAIDS development; some are susceptible (e.g., C57BL/6), while others are resistant (e.g., CBA and B10.BR). As an early block to viral replication in resistant mice has been demonstrated previously by PCR studies, we postulated that alpha/beta interferons (IFN-alpha/beta) may be involved in resistance to MAIDS. Susceptible C57BL/6 mice infected with LP-BM5 were treated with IFN-alpha/beta or Newcastle disease virus. Newcastle disease virus induces high endogenous IFN-alpha/beta production in mice. Both treatments delayed the development of MAIDS, as assessed by splenomegaly and T- and B-cell proliferation. In addition, an IFN-alpha/beta response was detected by reverse transcription-PCR and dot blotting 3, 6, and 9 h after LP-BM5 infection in resistant mice but not in susceptible mice. These results suggest that the ability to produce IFN-alpha/beta in response to LP-BM5 infection may contribute to host resistance to MAIDS.

Amelioration of experimental systemic lupus erythematosus (SLE) by retrovirus infection.

Experimental SLE can be induced in susceptible 129/J mice by immunization with a human anti-DNA antibody bearing a common idiotype designated 16/6 Id. Immunized mice develop autoantibodies, leukopenia, proteinuria, and immune complex deposits in renal glomeruli. Case reports have described clinical improvement in SLE in individuals becoming infected with HIV-1. Because 129/J mice are susceptible to experimental SLE and to infection with the BM5def murine leukemia virus (MuLV) mixture but do not develop the lymphoproliferative/ immunodeficiency disorder known as murine AIDS (MAIDS), we superimposed this infection on immunization with the 16/6 Id. Multiple effects were observed. First, we noted an amelioration in the course of experimental SLE. Second, both in experimental SLE and in BM5def MuLV infection, immunoreactivity to HIV-1 gp120 was demonstrated, although gp120 is not present in the BM5def MuLV viruses. Third, production of autoantibodies characteristically found in SLE, e.g., anti-DNA, anti-RNP, and anti-SSA, was seen in BM5def MuLV-infected mice, demonstrating that an immune response as a consequence of infection had occurred despite the absence of MAIDS induction. We conclude that (1) retrovirus inoculation may ameliorate the course of experimental SLE; and (2) retrovirus inoculation, even in the absence of MAIDS induction, induces an immunologic response which promotes the production of potentially pathogenic autoantibodies.

Resistance to murine AIDS in offspring of mice infected with LP-BM5. Role of CD8 T cells.

The murine-acquired immunodeficiency syndrome (MAIDS) is caused by a mixture of murine leukemia viruses (LP-BM5 MuLV). The influence of perinatal contact with retroviruses or their Ags on the response to infection was tested by infecting with LP-BM5 (MuLV) the adult offspring of mice with MAIDS. These offspring were resistant to disease after virus challenge. Most of them were free of defective viral DNA, and even those with molecular evidence of infection had lymphoid cells with a lower infectious capacity to cause MAIDS in naive recipients. No ecotropic, xenotropic, or mink cell focus-forming (MCF) virus expression was found at the age of 5 wk, which is the time of LP-BM5 (MuLV) challenge. However, at 22 wk of age, one-half of the offspring from MAIDS mothers had ecotropic virus-expressing cells in their spleens. At the time of suckling, offspring from infected mothers had enhanced percentages of B cells and CD4 and CD8 T cells in the spleen, possibly followed by a slight persistent splenomegaly. These results suggest that immune reactivity, rather than tolerance to the virus, is responsible for resistance to disease after challenge. The offspring of MAIDS mice could clear the virus after challenge. This clearance was mediated by CD8 T cells, as continuous CD8 T cell depletion initiated at the time of viral challenge abrogated the resistance of these mice to MAIDS.

Inhibition of Murine AIDS by Combination of AZT and Dideoxycytidine 5'-Triphosphate

A combination of antiretroviral drugs acting on different cell types (lymphocytes and macrophages) was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). In a first experiment, C57BL/6 mice were infected with a single i.p. administration of LP-BM5 and treated with 0.125 or 0.25 mg/ml AZT in drinking water for 3 months. AZT treatment was found to reduce lymphadenopathy (60 and 65 percent, respectively), splenomegaly (37 and 50 percent, respectively), and hypergammaglobulinemia (6 and 50 percent, respectively). Furthermore, at the highest AZT concentration, BM5d proviral DNA content in lymph nodes and in the spleen showed a reduction of 78 and 70 percent, respectively, compared to untreated animals. In a second experiment, infected mice were treated with AZT (0.25 mg/ml in drinking water) and with 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes for macrophage protection. Combined treatments resulted in a further reduction of lymphadenopathy (a further 33 percent with respect to the single treatment of AZT) and splenomegaly (a further 28 percent respect to the single treatment of AZT) but not of gammaglobulinemia. Proviral DNA in lymph nodes and spleen showed a reduction of 82 and 77 percent, respectively, compared to infected mice. Stimulation index of T cells was also significantly increased in animals receiving both treatments versus AZT only. In conclusion, the selective administration of antiviral drugs that preferentially protect different cell types seems to provide additional advantages compared to single-agent therapy.