Effect of anti-interferon-γ monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains

Immunization with myelin basic protein (MBP) in complete Freund's adjuvant failed to induce experimental allergic encephalomyelitis (EAE) in six resistant mouse strains studied: A/J, BALB/c C3H/HeJ, AKR, NZW and DBA/2. However, treatment of challenged mice with anti-interferon-γ (IFN-γ) monoclonal antibody (mAb) induced severe EAE in mice of all strains except AKR. Furthermore, anti-IFN-γ mAb treatment led to increased disease incidence and severity in BALB/c mice challenged with the MBP peptide_87–103, known to be encephalitogenic for the susceptible SJL strain. In three strains tested, anti-IFN-γ mAb enhanced passively induced EAE in the A/J and C3H/HeJ but not in the BAlB/c mice. All mice with clinically overt EAE had widespread histological lesions characterized by mononuclear cell infiltrates and focal demyelination. The results indicate that resistant strains are genetically capable of developing EAE, and that IFN-γ can contribute to disease resistance.     

Treatment with Anti-interferon-γ Monoclonal Antibodies Modifies Experimental Autoimmune Encephalomyelitis in Interferon-γ Receptor Knockout Mice

The role of interferon-γ (IFN-γ) in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) is still controversial. We have studied the function of IFN-γ and its receptor in the EAE model using two different IFN-γ receptor knockout (IFN-γ R^−/−) mouse types: C57Bl/6×129Sv, with a disruption of the IFN-γ receptor cytoplasmic domain, and 129Sv, homozygous for a disrupted IFN-γ receptor gene. Mice were immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. A subgroup of mice was treated with anti-IFN-γ monoclonal antibodies (mAb) on day 8 postimmunization. Clinical scoring and both histological and immunohistochemical studies were undertaken for all groups. We hereby show that treatment with anti-IFN-γ mAb worsened the disease course of 129Sv wild-type mice. However, it decreased the mean daily score in IFN-γ R^−/− 129Sv and the incidence of the disease down to 50% in C57Bl/6×129Sv IFN-γ R^−/− mice. Moreover, after anti-IFN-γ mAb treatment, oxidative stress levels, metallothionein I and II antioxidant protein expression, and apoptoticneuronal death were increased in wild-type mice while decreased in IFN-γ R^−/− mice. These results suggest a putative alternative mechanism of action of this cytokine that works independent of its receptor.     

Effect of anti-interferon-gamma and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse.

SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN-gamma to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-gamma. These findings support the notion that IL-2 is required for disease induction whereas IFN-gamma plays a disease-limiting role early in the development of EAE.     

Alteration in the level of interferon-γ results in acceleration of Theiler's virus-induced demyelinating disease

Intracerebral (i.c.) inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. We examined the role of interferon (IFN)-γ in this virally induced pathogenesis. Intraperitoneal (i.p.) injection of susceptible mice with an IFN-γ neutralizing monoclonal antibody (mAb), DB-1, resulted in a significantly accelerated onset of disease. The anti-IFN-γ mAb-treated animals showed a strong delayed-type hypersensitivity (DTH) response to the virus similar to that of control mAb-treated animals. Treatment with anti-IFN-γ mAb appeared to decrease TMEV-specific mAb titers in one of the protocols used. Intracerebral injection of the anti-IFN-γ mAb had no significant effect on the clinical course of disease. However, intracerebral administration of recombinant IFN-γ significantly accelerated the onset of TMEV-induced disease, as well as enhanced TMEV-specific T cell proliferation and DTH responses. The enhancing effect of IFN-γ was completely abrogated by simultaneous treatment with anti-IFN-γ mAb. Collectively, our data suggest that the level of IF-γ plays a key role in the TMEV-induced inflammatory respopnse and a perturbation of this balance may result in an alteration in the course of the demyelinating disease.     

Endogenous gamma interferon produced in central nervous system by systemic infection infection with Theiler's virus in mice

Theiler's virus GD VII strain causes acute encephalomyelitis by intracerebral inoculation. We established acute encephalomyelitis in mice by the intravenous (i.v.) inoculation of Theiler's virus GD VII strain. Replication of Theiler's virus injected i.v. could be observed in both the brain and spinal cord of mice, and interferon (IFN)-γ could be detected in the extracts of brain and spinal cord in parallel with viral replication. Furthermore, by the injection of anti-IFN-γ monoclonal antibody (mAb) on Day 1 post-infection (p.i.), mortality and virus titres in the spinal cord increased compared with the control mice treated with normal rat globulin. The histological exacerbation of inflammation was observed in spinal cord of anti-IFN-γ mAb-treated mice. These results indicate that endogenous IFN-γ, produced locally in the brain and spinal cord of mice through both antiviral action and anti-inflammatory action of IFN-γ in central nervous system, plays an important role in Theiler's virus infection.     

Transforming growth factor-β1 inhibits tumor necrosis factor-α/lymphotoxin production and adoptive transfer of disease by effector cells of autoimmune encephalomyelitis

We previously reported that the CD4⁺ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-β (TGF-β). We also reported that TGF-β downregulates interferon-γ (IFN-γ), but not interleukin-2 (IL-2) production, by the CD4⁺ effector T cells (Te) that mediate EAE. We now report that TGF-β also inhibits the production of tumor necrosis factor/lymphotoxin (TNF/LT) by EAE effector cells. When activated in vitro with myelin basic protein (MBP), Te produced TNF/LT, as measured using a WEHI 164 cytotoxicity assay. The specificity of cytokine action was demonstrated using neutralizing antibodies to TNF/LT. When added to the Te + MBP cultures, TGF-β inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-β antibodies augmented TNF/LT production in the Te + MBP cultures. We also confirm that TGF-β inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-γ production by Te. We conclude that TGF-β production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-γ production by the effector cells that mediate EAE.     

Suppression of Autoimmune Neuritis in IFN-γ Receptor-Deficient Mice

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain–Barré syndrome. In this autoimmune inflammatory disease, CD4⁺ T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-γ are intimately involved in causing pathogenic effects. To investigate the role of IFN-γ in cell-mediated EAN, IFN-γ receptor-deficient mutant (IFN-γR^−/−) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180–199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-γR^−/− mice exhibited later onset of clinical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4-producing cells were found in sciatic nerve sections from IFN-γR^−/− mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-γR^−/− mice had less infiltration of inflammatory cells, including macrophages, CD4⁺ T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-γ-secreting cells from the spleen were significantly augmented in the IFN-γR^−/− mice, reflecting a failure of negative feedback circuits. The IFN-γR deficiency did not affect the production of anti-P0 peptide 180–199-specific antibodies. These results indicate that IFN-γ contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.     

In vivo and in vitro studies of the prevention of proteolipid apoprotein-induced murine experimental allergic encephalomyelitis by monoclonal antibody against L3T4

The suppressive effect of anti-L3T4 monoclonal antibody (mAb) on murine experimental allergic encephalomyelitis (EAE) induced by sensitization with proteolipid apoprotein (PLP) was examined in vivo and in vitro. This mAb inhibited the antigen-specific proliferation of the encephalitogenic T cell lines but did not block the mitogen-mediated response. Serial injections of the mAb during the pre-effector phase of EAE markedly suppressed the development and relapse of the disease but this treatment initiated after appearance of clinical signs was not effective. In treated animals, L3T4+ T cells in the spleen were profoundly decreased and the antigen-specific proliferative response of spleen cells was completely suppressed. Moreover, adoptive transfer of spleen cells from the treated mice induced resistance against EAE induction in the recipients. However, no obvious evidence for antigen-specific suppressor cells was found in vitro in the L3T4- populations of spleen cells from treated mice.     

Suppression of experimental autoimmune myasthenia gravis and experimental allergic encephalomyelitis by oral administration of acetylcholine receptor and myelin basic protein: double tolerance

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChR or MBP and complete Freund's adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChR + MBP + CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-γ secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-γ in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR + MBP + CFA alleviated clinical disease as well as AChR- and MBP-specific B cell responses and autoantigen-induced IFN-γ secretion and production, but upregulated antigen-induced TGF-β mRNA expression in lymph node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-γ and augmentation of TGF-β are pivotal in tolerance induction.     

Theiler's virus is eliminated by a gamma-interferon-independent mechanism in the brain

The intravenous infection of Theiler's virus GD VII strain causes acute encephalomyelitis in infected mice. To determine the cellular mechanism of resistance and interferon (IFN)-γ-producing cell populations, mononuclear cells isolated from tissues of the brain were analyzed by the flow cytometry method. Antibodies specific for CD3, CD4, CD8, T cell receptor (TCR)-αβ, and Asialo GM1 were used to deplete the corresponding cell populations in Theiler's virus-infected mice. CD4⁺ lymphocytes and CD8⁺ lymphocytes infiltrated in the brains of infected mice from 5 days postinfection (p.i.). The number of CD3⁺/TCR-γδ⁺ lymphocytes increased in the brains on Day 6 p.i. The elimination of CD3⁺ lymphocytes or CD4⁺ lymphocytes augmented viral replication and suppressed the production of IFN-γ. The suppression of IFN-γ production by anti-CD3 monoclonal antibody (mAb) persisted, although the suppression by anti-CD4 mAb was observed only on Day 6 p.i. The depletion of CD8⁺ lymphocytes as well as TCR-αβ⁺ lymphocytes also augmented the viral replication; however, it did not alter the production of IFN-γ. Anti-Asialo GM1 antibody had no effect on viral replication and IFN-γ production. These results indicate that T lymphocytes are important for eliminating Theiler's virus from the brain, CD3⁺/CD4⁺/CD8⁻ lymphocytes and CD3⁺/TCRαβ⁻/CD4⁻/CD8⁻ lymphocytes would produce IFN-γ in brain. However, from the result on the experiment of the depletion of TCR-αβ⁺ lymphocytes, the defence mechanisms by T lymphocytes against Theiler's virus would be independent of endogenous IFN-γ production.     

T cell immunity and interferon-γ secretion during experimental allergic encephalomyelitis in Lewis rats

An immunospot assay that detects single secretory cells was used to enumerate interferon-γ secreting cells (IFN-γ-sc) in mononuclear cell suspensions from the central nervous system (CNS) and peripheral lymphoid organs after actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. In the CNS compartment there was a significant increase in the number of IFN-γ-sc preceding the onset of the clinical signs of EAE. Both in rats with EAE and rats immunized with Freund's complete adjuvant (FCA) the number of IFN-γ-sc increased in peripheral lymphoid organs, as compared to non-immunized controls. In view of the potent immunoregulatory effects of IFN-γ, its intra-CNS secretion may play a crucial role for clinicophatological events in EAE.To study the numbers of primed T cells that in response to myelin antigens produced IFN-γ, mononuclear cell suspensions from peripheral lymphoid organs were precultured to allow for antigen uptake, presentation and T cell triggering, followed by enumeration of IFN-γ-sc. T cells responding to a peptide of myelin basic protein (MBP) that previously have been shown encephalitogenic in Lewis rats, appeared initially and were quantitatively dominant over the course of EAE. Later, T cell reactivities to multiple regions of MBP appeared, showing that the concept of immunodominance in EAE is non-absolute and time dependent.Splenocyte cultures from EAE rats exposed to the different antigens showed a reduced number of IFN-γ-sc compared to cultures not exposed to antigen, suggesting an antigwn-induced suppression of T cell effector molecules.     

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ_1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ_1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Correlation between susceptibility to demyelination and interferon-γ induction of major histocompatibility complex class II antigens on murine cerebrovascular endothelial cells

The induction of major histocompatibility complex (MHC) Class II expression was studied on cerebrovascular endothelial cells (CVE) obtained from strains of mice that are resistant (BALB/c) and susceptible (SJL and CBA) to Theiler's virus-induced demyelination (TVID). Following 24 h treatment with interferon (IFN)-γ, MHC Class II was induced on CVE derived from susceptible but not resistant strains of mice. However, IFN-γ induced the expression of MHC Class II on late passages of BALB/c CVE. These results demonstrate a correlation between susceptibility to demyelination and the ability of IFN-γ to induce the expression of MHC Class II on CVE. In susceptible strains of mice, the presence of activated, IFN-γ-secreting T cells, in the vicinity of CVE would increase the antigen-presenting capabilities of CVE and result in increased T cell traffic into the central nervous system.     

Sulfasalizine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells

Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicylic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-γ-secreting cells (IFN-γ-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-γ secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-γ-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-γ-sc method may be relevant for the evaluation of new immunotherapeutic strategies in flammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.     

Contrasting effects of anti-adhesion molecule therapy in experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis.

An augmentation of experimental allergic encephalomyelitis (EAE) was observed when monoclonal antibody (mAb) to intercellular adhesion molecule 1 (ICAM-1) was administered after adoptive transfer. Clinical disease was more severe in the ICAM-1 specific mAb-treated EAE mice and included prominent ataxia compared to the PBS-treated controls or Theiler's murine encephalomyelitis virus (TMEV) infected mice treated with ICAM-1 specific mAb. Neuropathologic evaluation demonstrated a distinctly different distribution of lesions in the anti-ICAM-1-treated EAE mice which featured prominent demyelination and inflammation in the cerebellum, brainstem and cerebrum. These structures were minimally involved in the control mice and mAb treatment did not alter the neuropathology in TMEV-infected mice. These results indicate that anti-ICAM-1 can alter trafficking of lymphocytes and mononuclear cells in EAE but not TMEV-induced demyelinating disease.     

Interferon-γ Induces Apoptosis and Augments the Expression of Fas and Fas Ligand by Microglia in Vitro

Activation of microglia by interferon-γ (IFN-γ) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-γ has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-γ-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-γ (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-γ concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-γ also potentiated the expression of Fas and FasL in a similar dose–response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-γ may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-γ modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes.