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.     

Suppression of autoimmune neuritis in IFN-gamma 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)-gamma are intimately involved in causing pathogenic effects. To investigate the role of IFN-gamma in cell-mediated EAN, IFN-gamma receptor-deficient mutant (IFN-gammaR(-/-)) 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-gammaR(-/-) 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-gammaR(-/-) mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-gammaR(-/-) 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-gamma-secreting cells from the spleen were significantly augmented in the IFN-gammaR(-/-) mice, reflecting a failure of negative feedback circuits. The IFN-gammaR deficiency did not affect the production of anti-P0 peptide 180-199-specific antibodies. These results indicate that IFN-gamma contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.     

CD28-B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) 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. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.     

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.     

Tumor necrosis factor-α in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system

This study reports on the immunocytochemical localization of tumor necrosis factor-alpha (TNFα) in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system (PNS) using teased nerve fiber preparations. In experimental autoimmune neuritis induced by active immunization (EAN) or by adoptive transfer of autoreactive T cells (AT-EAN), macrophages passing blood vessels as well as macrophages adherent to nerve fibers were TNFα-positive. Large post-phagocytic macrophages at later stages of demyelination were TNFα-negative. Intraperitoneal application of an anti-TNFα antibody to EAN rats significantly reduced the degree of inflammatory demyelination, suggesting a pathogenic role for TNFα. After nerve transection only macrophages located within degenerating nerve fibers were TNFα-positive, while those entering and leaving nerves were negative. TNFα produced by macrophages seems to bevolved in immune-mediated demyelination and non-immune myelin degradation after axotomy. While interferon-gamma (IFNγ) is present in EAN nerves and may act as a local stimulus for TNF expression, the nature of this signal in Wallerian degeneration in the absence of IFNγ is unknown.     

Dual role of B cells with accelerated onset but reduced disease activity in P0106–125-induced experimental autoimmune neuritis of IgH0/0 mice

The role of B cells in autoimmune-mediated diseases of the peripheral nervous system was studied in experimental autoimmune neuritis (EAN) in B cell deficient IgH^0/0 C57BL/6J mice having been immunized with P0_106–125 peptide. Compared to coisogenic IgH^+/+ mice, onset of EAN was accelerated [100% disease incidence at day 9 post immunization (p.i.) vs. day 15 p.i.]. At day 9 p.i., numbers of P0_106–125-specific interferon (IFN)-γ-producing CD4⁺ T cells were increased, while IL-10 mRNA and production were decreased in IgH^0/0 mice. Beyond day 9 p.i., declining disease activity and a significant reduction of maximal disease activity were correlated with significantly reduced numbers of IFN-γ-producing CD4⁺ T cells in IgH^0/0 mice as compared with IgH^+/+ mice. Correspondingly, neuropathology demonstrated only mild axonal damage, while demyelination and dying back axonopathy with spinal cord motor neuron apoptosis were absent. Thus, depending on the stage of EAN, B cells play a dual, i.e. suppressive and enhancing, role during induction and at height of EAN, respectively. The combined interaction of B cells as well as CD4⁺ and CD8⁺ T cells is required for the development of EAN.     

γ-Hydroxybutyric acid-induced absence seizures in GluR2 null mutant mice

In this electrophysiological study, we examined the susceptibility of GluR2 mutant null mice to absence seizures in comparison with wild-type controls. The prodrug of (GHB), γ-butyrolactone (GBL) was given systemically to induce the absence seizures. We also tested the severity and duration of the seizure activity in this model. The results showed that the latency from GBL administration to onset of seizure was significantly prolonged in GluR2^−/− mice when compared to GluR2^+/+ mice. The duration of spike-and-wave discharges (SWD) was also significantly decreased in the GluR2^−/− mice. Ninety minutes following GBL administration, wild-type animals continued to exhibit intermittent SWD bursts while GluR2^−/− mice had returned to baseline. These data suggest that the GluR2 subunit may be involved in the initiation and maintenance of absence seizures induced by GBL.     

Interferon-γ secretion by in vivo activated cytotoxic T lymphocytes from the blood and cerebrospinal fluid during mumps meningitis

Functional studies of cerebrospinal fluid T lymphocytes during acute viral infections of the nervous system are rare. Recently, we had the opportunity to investigate the requirments for interferon-γ (IFN-γ) production of human in vivo activated (primary) cytotoxic T lymphocytes (CTL) generated during acute viral meningitis. Two HLA-B7-restricted, CD4⁻, CD8⁺ CTL clones from cerebrospinal fluid of one patient with mumps meningitis were studied. Although lytic activity was restricted by HLA-B7, the clones produced similar amounts of IFN-γ when stimulated with HLA-matched and mismatched mumps virus-infected target cells. In addition, peripheral blood mononuclear cells of infected patients secreted significant amounts of IFN-γ when incubated with autologous or allogeneic (HLA-A/B-mismatched) mumps virus-infected target cells. T cells capable of lytic activity and IFN-γ secretion could only be isolated from venous blood during the initial phase of the infection. We suggest that the ability of human in vivo activated CTL to secrete INF-γ early during the course of inflammation and in a HLA-unrestricted fashion is important for the elimination of viruses invading the central nervous system.     

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.     

Quantitative autoradiography of μ-,δ- and κ1 opioid receptors in κ-opioid receptor knockout mice

Mice deficient in the κ-opioid receptor (KOR) gene have recently been developed by the technique of homologous recombination and shown to lack behavioural responses to the selective κ₁-receptor agonist U-50,488H. We have carried out quantitative autoradiography of μ-, δ- and κ₁ receptors in the brains of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) KOR knockout mice to determine if there is any compensatory expression of μ- and δ-receptor subtypes in mutant animals. Adjacent coronal sections were cut from the brains of +/+, +/− and −/− mice for the determination of binding of [³H]CI-977, [³H]DAMGO ( -Ala²-MePhe⁴-Gly-ol⁵ enkephalin) or [³H]DELT-I ( -Ala² deltorphin I) to κ₁-, μ- and δ-receptors, respectively. In +/− mice there was a decrease in [³H]CI-977 binding of approximately 50% whilst no κ₁-receptors could be detected in any brain region of homozygous animals confirming the successful disruption of the KOR gene. There were no major changes in the number or distribution of μ- or δ-receptors in any brain region of mutant mice. There were, however some non-cortical regions where a small up-regulation of δ-receptors was observed in contrast to an opposing down-regulation of δ-receptors evident in μ-knockout brains. This effect was most notable in the nucleus accumbens and the vertical limb of the diagonal band, and suggests there may be functional interactions between μ- and δ-receptors and κ₁- and δ-receptors in mouse brain.     

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.     

Axonal transport of α-bungarotoxin binding sites in rat sciatic nerve

[¹²⁵I]α-Bungarotoxin (α-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [¹²⁵I]α-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [¹²⁵I]α-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature.d-Tubocurarine, nicotine, decamethonium and atropine displaced [¹²⁵I]α-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [¹²⁵I]α-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.     

Neurotrophin-3 deficient Schwann cells impair nerve regeneration

Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell (SC) survival and differentiation in the absence of axons. Prior studies have failed to define the explicit role of SC versus axon in NT-3 deficiency in relation to nerve regeneration and associated remyelination. In the paradigm we studied, using NT-3 heterozygous (NT3^+/−) knockout mice capable of survival into adult-life, the experimental design provided a model uniquely capable of differentiating SC/axon influences. In these studies we first identified a defect in nerve regeneration characterized by fewer SCs in the regenerating nerve fibers of crushed sciatic nerves of NT3^+/− mice. Subsequent experiments differentiated SC versus axonal influences as the culprit in defective nerve regeneration using sciatic nerve transplant paradigms. Results show an impairment in nerve regeneration in NT3^+/− mice with a retardation of the myelination process, and this defect is associated with decreased SC survival and an increase in the neurofilament packing density of regenerating axons. These observations indicate that NT3^+/− status of the SCs, but not of the axons, is responsible for impaired nerve regeneration and that NT-3 is essential for SC survival in early stages of regeneration-associated myelination in the adult peripheral nerve.     

A study of associated cell-mediated immune mechanisms in experimental autoimmune neuritis rats

The aims of this study are to investigate the optimal antigens used to induce acute or chronic EAN and the associated cell-mediated immune mechanisms. Lewis rats were grouped into EAN rats and control rats. EAN rats were immunized by injection into both hind footpads of inoculums containing 100 microg/200 microg of P2 peptide 57-81 and FCA, or 200 mug of P0 peptide 180-199 and FCA. Control rats were immunized by FCA. Clinical scores were compared at the maximum of disease. On the 14th day after immunization, we examined lymphocyte proliferation, fractions of CD4+ T cells within lymph node mononuclear cells (MNC), frequencies of CD4+CD25+ T cells within CD4+ T cells, supernatant productions of IFN-gamma, IL-4, IL-10 and TGF-beta1 secreted by lymphocytes. Histopathology of sciatic nerves was assessed. Our findings indicated: (1) 100 microg of P2 peptide 57-81 and 200 microg of P0 peptide 180-199 may induce an acute EAN and 200 microg of P2 peptide 57-81 may induce a chronic EAN; (2) Lewis rats were more sensitive to P2 peptide 57-81 than P0 peptide 180-199; (3) clinical disease had nothing to do with a change of relative CD4+ T cells number in lymph node MNC; (4) frequencies of CD4+CD25+ T cells and levels of TGF-beta1 secreted by lymphocytes negatively paralleled clinical EAN, while levels of IFN-gamma secreted by lymphocytes roughly paralleled clinical EAN at the acute phase; (5) sciatic nerve sections from the chronic EAN rats didn't show any inflammatory cells, but showed remaining segmental demyelination and axonal collapse at the chronic phase; (6) self-limitation of acute EAN may owe to the rising levels of IL-4 and IL-10, while a longer duration of chronic EAN may owe to the decreasing levels of IL-4 and IL-10.     

Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB₁ receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D₃ receptor null (D3R^(−/−)) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R^(−/−) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R^(−/−) mice also exhibited different endocannabinoid and TRPV1, but not CB₁, levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB₁ and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R^(−/−) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.     

Osteopontin is extensively expressed by macrophages following CNS demyelination but has a redundant role in remyelination

Osteopontin (OPN) is a key immunoregulator in the autoimmune-mediated demyelinating disease multiple sclerosis. OPN may also play a role in the remyelination since it is 1) a ligand for αV integrins, several of which regulate the properties of the oligodendrocyte precursor cells (OPCs) primarily responsible for remyelination, and 2) enhances myelin membrane formation in OPC lines. Here we show that OPN is expressed at high levels during remyelination of toxin-induced demyelination. The increased expression is due to mRNA expression in macrophages and follows differences in macrophage responses to demyelination in young and old adult animals. To identify the role of OPN in remyelination focal demyelination was induced in wild-type and OPN^−/− mice. There was no difference in the rate of remyelination between the two groups indicating that OPN is not a critical component of remyelination.     

CD4 and CD8 T cells,but not B cells,are critical to the control of murine experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune disease of the peripheral nervous system that duplicates the clinical, pathological, and electrophysiological features of Guillain-Barré syndrome in humans. However, the molecular pathogenesis of EAN remains controversial. Therefore, for this study, we induced EAN with P0 protein peptide 180-199 in CD4(-/-), CD8(-/-), CD4(-)8(-), and B cell knockout (microMT) mice to further investigate the roles of these cells in EAN. Our results showed that the severity of clinical signs and histopathological manifestations of EAN and the T cell response to P0 peptide 180-199 in CD4(-/-) mice were significantly lower than those in their wild-type counterparts. CD8(-/-) mice also had a milder clinical course, less histopathological change, and a diminished T cell response to P0 peptide 180-199. However, more severe clinical and histopathological manifestations, a stronger T cell response to P0 peptide 180-199, and enhanced IFN-gamma production in the spleen were observed in the EAN of CD4(-)8(-) and microMT mice, but these were not obviously different from those of wild-type mice. Levels of IgG production were similar in sera from CD4(-/-), CD8(-/-), and CD4(-)8(-), and wild-type mice. These findings suggest that the induction and control of murine EAN are dependent on both CD4(+) and CD8(+) T cells and that B cells apparently do not perpetuate the related inflammatory demyelination.