Further evidence for a role of nitric oxide in experimental allergic encephalomyelitis: aminoguanidine treatment modifies its clinical evolution

The role of nitric oxide (NO) in inflammatory/demyelinating diseases is undergoing extensive investigation as a potential target for therapeutic intervention. However, interference with NO production has resulted in contrasting effects on the development of experimental allergic encephalomyelitis (EAE), the most widely used experimental model for multiple sclerosis (MS). Purpose of this paper was both the analysis of the individual clinical evolution of EAE induced in Lewis female rats by active immunisation and the evaluation of the effect of treatment with aminoguanidine, a selective inhibitor for the inducible isoform of nitric oxide synthase (iNOS). In our experimental model, relapse occurred in 66% of animals. Aminoguanidine treatment, started 3 days before immunisation, guaranteed a complete recovery from the acute phase and a delayed, milder relapse. Moreover, 79 days after immunisation inflammatory cellular infiltrates in the spinal cord were reduced. These data further support the involvement of NO in EAE evolution.     

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS). which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some infiltrating inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.     

Immunologic differences in murine glial cells and their association with susceptibility to experimental allergic encephalomyelitis

We tested the hypothesis that glial cells from mice resistant or susceptible to the autoimmune disease experimental allergic encephalomyelitis (EAE) may differ in their abilities either to express Ia antigens and/or stimulate anti-class II (Ia)-specific T-cells. Ia antigens were induced on glial cells from EAE-susceptible (SJL/J) and -resistant (B10.S and DBA/2) strains of mice by culture with lymphokines from activated T-cells (2 degrees SN). Ia antigen expression was quantified with an enzyme-linked immunosorbent assay (ELISA) in which glia were exposed to monoclonal anti-Ia antibodies and alkaline phosphatase-labeled anti-mouse Ig antibodies. The ability of glial cells to stimulate anti-Ia T-cells was quantified by culturing irradiated glial cells with anti-Ia-specific T-cell lines and measuring the amounts of [3H]thymidine incorporated by these lines. Glial cells from all strains of mice could be induced to express Ia antigens and upon exposure to high concentrations of lymphokines, amounts of expressed Ia antigen were equivalent. However, at limiting lymphokine concentrations, glia from the EAE-resistant strain B10.S expressed greater amounts of Ia antigen than did glia from SJL/J mice (p less than 0.05), suggesting that B10.S glia were more sensitive to the Ia-inducing effects of T cell lymphokines. In contrast to the above results, glia from EAE-susceptible SJL mice consistently demonstrated an increased ability to induce T-cell proliferation in lines specific for Ias antigen, compared to glia from EAE-resistant mice, even those of the same Ia haplotype (i.e. B10.S). Spleen cells from resistant strains had equivalent and frequently greater ability to induce anti-Ia-specific T-cell proliferation than did SJL spleen cells. These data suggest (a) that there are differences in the sensitivity of glia from different strains of mice to the Ia antigen-inducing effects of T-cell lymphokines, (b) that expression of Ia antigen does not necessarily correlate with the ability to stimulate Ia-specific T-cells, (c) that there are organ-specific differences in the ability to stimulate Ia antigen-specific T-cells, and (d) that an additional variable involved in determining resistance or susceptibility to an organ-specific autoimmune disease may be the ability of the target organ to stimulate anti-Ia-specific T-cells.     

Our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review

Nitric oxide was first described being produced in inflammatory cells involved in experimental autoimmune encephalomyelitis in 1992. Since then some 45 papers have appeared examining the role of NO in this central nervous system autoimmune inflammatory disease. Of the first 10 papers published all resulted in the interpretation that NO was a pathologic or “bad” molecule in the context of EAE. A few papers then began to appear suggesting that NO may not in fact always be a harmful molecule and by the end of 1997 early 1998, 22 papers suggested a destructive role for the molecule while three suggested it was protective. The past two years have seen a significant increase in reports supporting a protective mechanism for NO in EAE such that as of July 1999, 27 papers suggest a destructive and 15 a protective role for NO with a few uncommitted. This review sets out in a more or less chronological order the studies examining the role of NO in EAE and maps our changing understanding of the molecules role in this CNS inflammatory disease and by inference perhaps multiple sclerosis.     

基质金属蛋白酶9在实验性自身免疫性脑脊髓炎大鼠发病过程中的动态表达

目的通过检测基质金属蛋白酶-9(MMP-9)在EAE大鼠发病过程中不同阶段外周血和中枢神经系统中的表达水平及动态变化,以探讨其在多发性硬化发病过程中的作用及机制。方法 Wistar雌性大鼠80只,分为模型组(EAE组)、完全福氏佐剂组(CFA组),分别于免疫后6天、8天、10天、12天、14天、16天、18天及20天取视交叉处脑组织和脊髓腰膨大段行HE染色观察炎性细胞的浸润状况;免疫组化法检测脑和脊髓组织中MMP-9的表达,酶联免疫吸附实验测定血清中MMP-9的含量。结果 EAE组大鼠脊髓和脑组织中MMP-9阳性细胞数及血清中MMP-9水平均显著高于同期CFA组(P<0.05);EAE 10天组与EAE 6天、14天、18天及20天组比较,大鼠脊髓腰膨大处MMP-9阳性细胞数较多(P<0.05);EAE 12天组与EAE 6天组、8天组、14天组、16天组、18天组及20天组比较,大鼠脑组织视交叉处MMP-9阳性细胞数较多(P<0.05);EAE 12天组与EAE 6天组、8天组、10天组、14天组、16天组、18天组及20天组相比较,大鼠血清中MMP-9含量较高(P<0.05)。结论在EAE大鼠发病前期即有中枢组织内MMP-9的高表达,且EAE大鼠脊髓中MMP-9的高表达要早于脑组织和外周血,但MMP-9表达的高峰在第12天与病理变化和疾病进展是同步的。     

左旋咪唑诱导大鼠急性EAE外周血CD4+/CD8+和IL-6的检测及意义

目的 探讨急性实验性变态反应性脑脊髓炎(EAE)大鼠外周血CD4 /CD8 及IL-6的改变及意义.方法 采用豚鼠髓鞘蛋白匀浆和福氏完全佐剂注射并加用左旋咪唑诱发大鼠EAE模型,分别注射生理盐水/福氏佐剂、左旋咪唑作为对照,注射后16d采用Loyez氏髓鞘染色观察大鼠脊髓髓鞘改变、流式细胞仪检测外周血CD4 /CD8 比值和采用ELISA法检测IL-6水平,并与对照组比较.结果 模型组发病率87.5%,正常组、NS/CFA组和LMS对照组未见异常.模型组髓鞘染色见髓鞘结构疏松、部分髓鞘脱失;外周血CD4 /CD8 比值为2.33±1.04,及IL-6为194.92±56.85,较其余各组明显增高(P＜0.01,P＜0.01).结论 外周血CD4 /CD8 比值及IL-6水平增高在大鼠EAE的发生过程中发挥重要作用.     

Evidence for the production of peroxynitrite in inflammatory CNS demyelination

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.     

大鼠实验性变态反应性脑脊髓炎中一氧化氮的变化

目的　观察外周和中枢一氧化氮 (NO)在大鼠实验性变态反应性脑脊髓炎 (EAE)中的动态变化 ,探讨EAE大鼠发病的相关生物学机制。方法　 采用免疫组化法和硝酸还原酶法 ,观察豚鼠全脊髓匀浆诱导的Wistar大鼠EAE的过程中 ,脊髓内表达iNOS胶质细胞与外周NO代谢物NO 2 和NO 3的变化。 结果　 对照组脊髓内未发现表达iNOS阳性细胞 ,表达iNOS的CNS胶质细胞可能是小胶质细胞 ,而且它的变化与EAE大鼠的病情一致 ,评分 2分和 3分EAE大鼠脊髓表达iNOS的小胶质细胞比评分 1分大鼠明显增多 (P <0 .0 1) ,恢复期EAE大鼠表达iNOS的小胶质细胞明显减少 (P <0 .0 1)。EAE大鼠外周血清NO值随症状程度加重而升高 ,但在EAE恢复期时仍保持较高水平。未发病大鼠血清NO值明显增高 ,与对照组之间具有显著性差异 (P <0 .0 1)。 结论　 小胶质细胞产生的NO可能在急性期EAE大鼠的发病中起重要作用     

Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation

Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG_35-55/CFA and 100–600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.     

实验性过敏性脑脊髓炎豚鼠中枢神经系统一氧化氮合酶的表达

目的探讨诱导型一氧化氮合酶（ｉＮＯＳ）在中枢神经系统脱髓鞘疾病中的作用。方法采用硫辛胺脱氢酶染色和抗诱导型一氧化氮合酶（抗ｉＮＯＳ）抗体的免疫组化方法，对髓鞘碱性蛋白诱导豚鼠产生的实验性过敏性脑脊髓炎（ＥＡＥ）病程中，脑和脊髓的一氧化氮合酶（ＮＯＳ）和ｉＮＯＳ表达情况进行研究。结果在ＥＡＥ的急性期主要为血管、血管周围细胞、浸润细胞和小胶质细胞显示ｉＮＯＳ免疫反应阳性，在恢复期星形细胞则出现免疫反应阳性。结论提示一氧化氮是ＥＡＥ早期血脑屏障破坏以及进展期髓鞘和少突胶质细胞破坏的重要介导物质。     

Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-α), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-α suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN-γ secretion in both donors and recipients. IFN-α acts orally by modifying the encephalitogenicity of donor spleen T cells.     

Enriched environment regulates thymocyte development and alleviates experimental autoimmune encephalomyelitis in mice

Environmental and social factors have profound impacts on immune homeostasis. Our work on environmental enrichment (EE) has revealed a novel anti-obesity and anticancer phenotype associated with enhanced activity of CD8⁺ cytotoxic T lymphocytes in secondary lymphoid tissues. Here we investigated how an EE modulated thymus and thymocyte development. EE decreased thymus mass and cellularity, decreased the double positive thymocyte population, increased the proportion of CD8⁺ T cells, reduced the CD4:CD8 ratio, and downregulated CD69 expression in T cells. In a model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE), EE alleviated symptoms, inhibited spinal cord inflammation through regulation of type 1 T-helper cells mediated by glucocorticoid receptor signaling, and prevented EAE-induced thymic disturbance. Our mechanistic studies demonstrated that hypothalamic BDNF activated a hypothalamic-pituitary-adrenal axis mediating the EE’s thymic effects. Our results indicate that a lifestyle intervention links the nervous, endocrine, and adaptive immune system, allowing the body to adapt to internal and external environments.     

Analysis of gene expression in MOG-induced experimental autoimmune encephalomyelitis after treatment with a novel brain-penetrating antioxidant

Accumulating data from experimental studies indicate that oxidative stress has a major role in the pathogenesis of multiple sclerosis (MS). It has been suggested that local production of reactive oxygen species, probably by macrophages, mediates axonal damage in both MS patients and the mouse model experimental autoimmune encephalomyelitis (EAE). We have shown previously that our novel brain-penetrating antioxidant, N-acetylcysteine amide (AD4), reduces the clinical and pathological symptoms, including inflammation and axonal damage in myelin oligodendrocyte glycoprotein (MOG)-induced chronic EAE in mice. The aim of this study was to examine the molecular mechanism by which AD4 exerts protection in MOG-induced EAE mice. Therefore, we analyzed gene-expression profile in the spinal cords of MOG-induced chronic EAE mice and compared them with MOG-induced mice treated with AD4, using a cDNA microarray. We found that MOG treatment up-regulated genes encoding growth factors, cytokines, death receptors, proteases, and myelin structure proteins, whereas MOG- and AD4-treated mice demonstrated gene expression profiles similar to that seen in na?ve healthy mice. In conclusion, our study shows that chronic AD4 administration suppresses the induction of various pathological pathways that play a role in EAE and probably in MS.     

Clonal diversity of basic protein specific T cells in Lewis rats recovered from experimental autoimmune encephalomyelitis

T cell lines selected from Lewis rats recovered from experimental autoimmune encephalomyelitis (EAE) respond not only to the immunodominant 72-89 epitope of basic protein (BP), but also to secondary epitopes including the I-A restricted 43-67 region of guinea pig (Gp) BP and the I-E restricted 87-99 sequence of rat (Rt) BP. The current study demonstrates at the clonal level the diversity of T cell responses to Gp- and Rt-BP in EAE-recovered rats. As predicted from the response pattern of BP-selected T cell lines, T cell clones from the lines responded to both the dominant and secondary epitopes of BP. In addition, a new majority clonal type was identified that responded to whole BP but not to epitopes represented on enzymatic cleavage fragments or synthetic peptides spanning the BP molecule. Clones representative of each of the three types of Gp-BP responses were characterized for phenotype, major histocompatibility complex restriction, and biologic activity in vivo. All of the clones were strongly CD4+ and co-expressed CD8 at modest levels as measured by both immunofluorescence and Northern blots. All three T cell specificities were I-A restricted. However, only the 72-89 responsive clone could transfer clinical EAE, due most likely to its unique ability to respond to Rt-BP. In contrast, the Gp-BP 43-67 reactive T cell clone transferred protection against EAE, whereas the whole Gp-BP reactive clone transferred delayed-type hypersensitivity response but was neither encephalitogenic nor protective. Thus, the recovery process from EAE is distinguished by an increased diversity of protective clones as well as innocuous clones that may be spawned as encephalitogenic T cells are regulated.     

Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system

Gliosis is a repair process of lesions appearing in the central nervous system (CNS). Although gliosis by astrocytes (astrocytic gliosis) has been well documented, that by microglia (microglial gliosis) remains poorly understood. In the present study we induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats and examined microglial and astroglial reactions to EAE lesions at various stages of the disease by immunohistochemistry. For the demonstration of microglia and astrocytes, antibodies against complement receptor type 3 (OX42) and glial fibrillary acidic protein (GFAP) were used, respectively. It was revealed that the whole course of microglial and astroglial reactions to EAE lesions is divisible into three stages, i.e., initial, peak and recovery stages. Microglial and astroglial reactions to EAE lesions at each stage correspond well with the clinical and histological stages of EAE. At the initial stage, rats showed mild clinical signs and a few inflammatory foci were found in the CNS. Microglia were increased in number in close association with inflammatory cell aggregates, whereas astrocytes showed no significant reaction in spite of the presence of inflammatory cells. At the peak stage, rats showed full-blown EAE and the number of inflammatory cells reached maximum. The most characteristic finding at this stage was 'encasement' of inflammatory lesions by astrocytic fibers. Microglia were increased in number, but association of microglia with lesions was prevented by astrocytes. Interestingly, however, such characteristic distribution of microglia and astrocytes was not observed at the recovery stage. Residual inflammatory cell aggregates were intermingled with dense microglial and astrocytic gliosis, forming 'micro-astroglial scars'. Double immunofluorescence staining with anti-GFAP and anti-bromodeoxyuridine (BrdU), or with OX42 and anti-BrdU revealed that BrdU-incorporated microglia, but not astrocytes, were present mainly at the initial and peak stages, suggesting that microglia would proliferate by cell division to create gliosis, whereas astrocytic gliosis would be a result of migration of astrocytes and/or up-regulation of expression of GFAP molecule. Taken together with previous in vitro findings that microglia, but not astrocytes, stimulate encephalitogenic T cell proliferation, these in vivo findings suggest that microglia augment, whereas astrocytes suppress, inflammatory processes in the CNS.     

Induction of experimental autoimmune encephalomyelitis in Lewis rats by a viral peptide with limited homology to myelin basic protein

Viral infections are thought to play an important role in the pathogenesis of multiple sclerosis potentially through molecular mimicry, but direct evidence from humans and animal models remains inadequate. Based on the fact that amino acid homology has been found between viral and host encephalitogenic protein, we designed four viral peptides (peptides of HBV polymerase protein, large T protein of JC virus, EB virus DNA polymerase and alkaline exonuclease of Human herpesvirus 6) with limited homology to myelin basic protein and explored their clinical, immunological and histological characteristics in Lewis rats. The immunization with JC virus peptide induced slight clinical signs of EAE in Lewis rats. Immunological examination indicated that rats immunized with JC virus peptide triggered T-cell cross-reactivity against MBP68-86, but failed to induce antibody cross-reactivity with MBP68-86. Histological staining exhibited the infiltration of inflammatory T cells and the activation of microglia in spinal cords of rats immunized with MBP68-86 and JC virus peptide. Other three peptides had negative findings in Lewis rats. These results suggested that molecular mimicry could be an important factor in the pathogenesis of EAE induced with JC virus peptide by expanding a population of reactive T cells that recognize MBP68-86 in Lewis rats inferring a possible pathogenesis for molecular mimicry in MS.     

Chronic relapsing experimental autoimmune encephalomyelitis: Treatment with combinations of myelin components promotes clinical and structural recovery

Preliminary results are presented on the treatment of Strain 13 guinea pigs with chronic relapsing experimental autoimmune (allergic) encephalomyelitis (EAE) induced by a single sensitisation with whole spinal cord. Animals were treated at different stages of the disease with injections containing either myelin basic protein (MBP) alone in incomplete Freund's adjuvant (IFA), or MBP in combination with a lipid hapten of myelin, galactocerebroside (GC) in IFA. The rationale for this treatment stemmed from previous work which suggested that MBP was responsible for T cell sensitisation in EAE and that GC was important in producing demyelinating antibodies and that both myelin components were needed in the induction of disease. Although treatment with MBP alone caused some initial stabilisation of the disease process, subsequent relapses occurred in all animals. However, in animals given MBP and GC together, either early or late in the course of the disease, marked clinical improvement has been noted with little or no development of relapses over an observation period of more than one year posttreatment. In addition, evidence of extensive remyelination and oligodendroglial proliferation in CNS lesions has been found in MBP-GC-treated animals suggesting that this therapy might be beneficial for CNS repair and relevant to multiple sclerosis.