Effect of levamisole and etimisole on the development of experimental allergic encephalomeylitis

A single subcutaneous inoculation with 0.02 mg of heterologous myelin basic protein (MBP) in combination with Freund's complete adjuvant resulted in clinical and histological manifestations of experimental allergic encephalomyelitis (EAE) in 80-90% of treated guinea pigs. Daily parenteral administration of levamisole and etimisole during the latent period produced a suppressive effect on EAE development, reducing morbidity and mortality rates and preventing pathomorphological changes in the CNS. Animals receiving the drugs had decreased delayed hypersensitivity reactions to MBP in vitro. Etimisole brought about a moderate decline in the formation of circulating anti-MBP antibodies, while levamisole did not affect the strength of the humoral response, something which confirmed the primary role of cell-mediated immune reactions in the CNS demyelinization process. The reported findings may be significant in developing therapeutic strategies with respect to demyelinization diseases.     

Cerebrospinal fluid and serum oligoclonal IgG bands in rabbits with experimental allergic encephalomyelitis

Rabbits sensitized with whole nervous tissue or myelin basic protein (MBP) plus adjuvant and developing experimental allergic encephalomyelitis (EAE) were studied for the presence of oligoclonal immunoglobulin (Ig) bands in spinal fluid and serum. Samples obtained prior to sensitization and at the time of sacrifice were concentrated and subjected to agar gel electrophoresis. Of 11 rabbits receiving whole nervous tissue and developing severe clinical signs of EAE, 7 showed new oligoclonal Ig bands in spinal fluid and in serum obtained 19 days or more after sensitization. With MBP sensitization, 2 of 6 rabbits exhibited new spinal fluid bands, while all 6 rabbits studied demonstrated serum banding. The bands were identified as IgG by immunochemical studies using peroxidase-labeled antisera and byStaph. protein A absorption. The majority of animals showed no banding in presensitization samples. The finding of oligoclonal IgG in EAE reveals yet another immunologic correlation between EAE and the human demyelinating disease, multiple sclerosis.     

Passive Experimental Autoimmune Encephalomyelitis in C57BL/6 with MOG: Evidence of Involvement of B Cells

Experimental autoimmune encephalomyelitis (EAE) is the most relevant animal model to study demyelinating diseases such as multiple sclerosis. EAE can be induced by active (active EAE) or passive (at-EAE) transfer of activated T cells in several species and strains of rodents. However, histological features of at-EAE model in C57BL/6 are poorly described. The aim of this study was to characterize the neuroinflammatory and neurodegenerative responses of at-EAE in C57BL/6 mice by histological techniques and compare them with that observed in the active EAE model. To develop the at-EAE, splenocytes from active EAE female mice were harvested and cultured in presence of MOG_35–55 and IL-12, and then injected intraperitoneally in recipient female C57BL6/J mice. In both models, the development of EAE was similar except for starting before the onset of symptoms and presenting a higher EAE cumulative score in the at-EAE model. Spinal cord histological examination revealed an increased glial activation as well as more extensive demyelinating areas in the at-EAE than in the active EAE model. Although inflammatory infiltrates composed by macrophages and T lymphocytes were found in the spinal cord and brain of both models, B lymphocytes were significantly increased in the at-EAE model. The co-localization of these B cells with IgG and their predominant distribution in areas of demyelination would suggest that IgG-secreting B cells are involved in the neurodegenerative processes associated with at-EAE.     

Inflammation in EAE: Role of chemokine/cytokine expression by resident and infiltrating cells

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) which has many clinical and pathological features in common with multiple sclerosis (MS). Comparison of the histopathology of EAE and MS reveals a close similarity suggesting that these two diseases share common pathogenetic mechanisms. Immunologic processes are widely accepted to contribute to the initiation and continuation of the diseases and recent studies have indicated that microglia, astrocytes and the infiltrating immune cells have separate roles in the pathogenesis of the MS lesion (1,2). The role of cytokines as important regulatory elements in these immune processes has been well established in EAE and the presence of cytokines in cells at the edge of MS lesions has also been observed (3–7). However, the role of chemokines in the initial inflammatory process as well as in the unique demyelinating event associated with MS and EAE has only recently been examined. A few studies have detected the transient presence of selected chemokines at the earliest sign of leukocyte infiltration of CNS tissue and have suggested astrocytes as their cellular source (8–10). Based on these studies, chemokines have been postulated as a promising target for future therapy of CNS inflammation. This review summarizes the events that occur during the inflammatory process in EAE and discusses the roles of cytokine and chemokine expression by the resident and infiltrating cells participating in the process. Special issue dedicated to Dr. Marion E. Smith.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Myelin lipophilin-induced demyelinating disease of the central nervous system

Purified lipophilin, a hydrophobic lipoprotein of myelin, induces a cell-mediated demyelinating disease of the central nervous system similar to experimental allergic encephalomyelitis (EAE) induced by the myelin basic protein (MBP). Guinea pigs challenged with lipophilin (emulsified with CFA) developed clinical and histological signs of disease indistinguishable from those developed by animals similarly challenged with MBP. Both lipophilin and MBP induced and elicited delayed-type hypersensitivity in animals challenged with respective antigens. Tryptophan, an essential component of the MBP-determinant for disease in guinea pigs, is required for the encephalitogenicity of lipophilin.     

Studies on autoimmune encephalomyelitis in the guinea pig. II. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor.

Complement-dependent demyelinating activity of whole brain homogenate (WBH)-induced experimental allergic encephalomyelitis (EAE) sera was tested on long term tissue cultures of in vitro myelinated fetal guinea pig cerebellum. Complement-fixing (CF) auto-antibodies were shown to be the responsible agents, as demonstrated in experiments where all reagents belonged to the same species: guinea pigs of outbred (Hartley) and even of inbred (S2 or S13) strains. These antibodies were of the IgG2 class as shown by Sephadex G-200 and DEAE cellulose fractionation experiments. The corresponding auto-antigen was present in the homogenate and myelin of the central nervous system (CNS) tissue. It was different from the encephalitogenic basic protein of CNS myelin (BP), as shown in experiments where the demyelinating auto-antibodies were induced, detected, and absorbed by WBH or by CNS myelin but not by BP. They were neither induced by nor cross-reacting with cerebroside and peripheral nervous system (PNS) tissue.     

Animal models for autoimmune demyelinating disorders of the nervous system

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that takes a relapsing-remitting or a progressive course (reviewed in Refs 1,2). Its counterpart in the peripheral nervous system (PNS) is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (reviewed in Ref. 3). In addition, there are acute, monophasic disorders, such as the inflammatory demyelinating polyradiculoneuropathy termed Guillain-Barré syndrome (GBS) in the PNS, and acute disseminated encephalomyelitis (ADEM) in the CNS. Both MS and GBS are heterogeneous syndromes. In MS different exogenous assaults together with genetic factors can result in a disease course that finally fulfils the diagnostic criteria. In both diseases, axonal damage can add to a primarily demyelinating lesion and cause permanent neurological deficits. No single animal model exists that mimics all the features of human demyelinating diseases; rather, the available models reflect specific facets. Here, we focus on experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN) as models in rat and mouse strains, and discuss their distinct histopathology and the roles played by different autoantigens.     

The immunopathology of chronic experimental allergic encephalomyelitis induced in rabbits with bovine proteolipid protein

The role of myelin proteolipid apoprotein (PLP) in the central nervous system (CNS) immune response of rabbits has been investigated by analyzing the immunopathology of chronic experimental allergic encephalomyelitis (EAE) induced by sensitization with PLP. Clinical disease occurred in seven out of nine rabbits sensitized with bovine PLP and monitored for up to 6 mo. Positive delayed hypersensitivity skin test reactions to PLP occurred in all but one of the PLP-sensitized animals. All PLP-sensitized animals had meningeal and CNS parenchymal inflammation that correlated with disease severity. Serial blood samples were stained with a panel of antibodies to rabbit T and B cells, as well as Ia, and large and small mononuclear cell populations were analyzed by flow cytometry. Peripheral leukocyte population staining did not correlate with clinical signs or sensitization to PLP. Cryostat CNS tissue sections were stained with the same set of antibodies by using an immunoperoxidase technique, and positive cells and vessels were counted. T cells and macrophages were numerous and in equal numbers in perivascular parenchymal inflammatory infiltrates, whereas B cells were less numerous (p less than 0.001). T cells also diffusely infiltrated the parenchyma. Most perivascular inflammatory cells and many scattered parenchymal cells were Ia+; Ia vascular expression was increased over controls (p less than 0.001), and also correlated with disease severity. The immunopathology of this chronic EAE model is the same as that of whole CNS tissue- and myelin basic protein-induced EAE in other species, and is similar to that of multiple sclerosis. Cellular immune responses to PLP may therefore contribute to systemic and in situ responses in CNS tissue demyelinating diseases.     

Mitochondria as possible "targets" in neuroallergy

During experimental allergic encephalomyelitic (EAE) in dogs, it was established with electron microscope that mitochondria of neurons and glia of the central nervous system (CNS) were most sensitive organelles to the development of neuroallergy. Swelling of mitochondria, disruption of their membranes, desorganization of mitochondrian cristae were observed in the incubation period; these alterations were seen increasing by the moment of clinical manifestation of EAE. Immunization of guinea pigs by mitochondrial fractions of dog's brain cortex with Freund's adjuvant resulted in the manifestation of clinical and morphological signs of EAE in the CNS in addition to a significant destruction of mitochondria. The idea of the initial alteration of different membranous structures of the CNS during demyelinating processes in brain is put forward.     

Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8(-/-) mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC(50) = 0.4 nM). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.     

Inosine,an Endogenous Purine Nucleoside,Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor

Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes.

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Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes were blocked by inosine treatment. Additionally, inosine consistently inhibited IL-17 levels in peripheral lymphoid tissue, as well as IL-4 levels and A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. EAE: experimental autoimmune encephalomyelitis; MS: multiple sclerosis; A2AR: adenosine A2A receptor; IL-17: interleukin-17; IL-4: interleukin-4

     

Cutting edge: C3, a key component of complement activation, is not required for the development of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in mice

Experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, is regarded as an experimental model for multiple sclerosis. The complement has been implicated in the pathogenesis of multiple sclerosis. To clarify the role of C in mouse EAE, we immunized mice deficient in C3 (C3(-/-)) and their wild-type (C3(+/+)) littermates with myelin oligodendrocyte glycoprotein peptide 35-55. C3(-/-) mice were susceptible to EAE as much as the C3(+/+) mice were. No differences were found for the production of IL-2, IL-4, IL-12, TNF-alpha, and IFN-gamma between C3(+/+) and C3(-/-) mice. This finding shows that C3, a key component in C activation, is not essential in myelin oligodendrocyte glycoprotein peptide-induced EAE in mice.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Location of a new encephalitogenic epitope (residues 43 to 64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL x PL)F1 mice.

Synthetic peptides of proteolipid protein (PLP) were screened for their ability to induce experimental autoimmune encephalomyelitis (EAE) in SJL/J, PL/J, and (SJL x PL)F1 mice, and T cell lines were selected by stimulation of lymph node cells with PLP peptides. PLP 141-151 was found to be less encephalitogenic in SJL/J mice than PLP 139-151, due to deletion of two amino acids from the amino-terminal end. PLP 139-151 immunization induced relapsing EAE in SJL/J and F1 mice but not PL/J mice. In contrast, PLP 43-64 induced relapsing EAE in PL/J and F1 mice but not SJL/J mice. F1 T cell lines specific for either PLP 43-64 or PLP 139-151 adoptively transferred demyelinating EAE to naive F1 recipients. Haplotypes H-2s and H-2u appear to be immunologically co-dominant in F1 mice in the PLP EAE system, which differs from the H-2u dominance in F1 mice in the myelin basic protein EAE system. The identification of a PLP peptide that is encephalitogenic in PL/J mice, in addition to the previous demonstration of PLP peptides that are encephalitogenic for SWR mice (PLP 103-116) and SJL/J mice (PLP 139-151), lends support to a role for PLP as a target Ag in autoimmune demyelinating diseases.     

Cytosolic phospholipase A2 plays a key role in the pathogenesis of multiple sclerosis-like disease

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that results in motor and sensory deficits. Although MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are thought to be T cell-mediated diseases, the mechanisms underlying the lesions in the CNS are not fully understood. We propose that a strong candidate as a central mediator in evoking the complex pathological changes seen in MS and EAE is the enzyme cytosolic phospholipase A2 (cPLA2). One of the metabolic products of this enzyme is pro-inflammatory, while the other induces myelin breakdown, demyelination, and chemokine/cytokine expression. We provide evidence that cPLA2 is highly expressed in EAE lesions and show that blocking this enzyme leads to a remarkable reduction in the onset and progression of EAE.     

Gene-Expression Profiling of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that serves as an experimental tool for studying the etiology, pathogenesis, as well as new therapeutic approaches of multiple sclerosis (MS). EAE is a polygenic chronic inflammatory demyelinating disease of the nervous system that involves the interaction between genetic and environmental factors. Previous studies have identified multiple quantitative trait loci (QTL) controlling different aspects of disease pathogenesis. However, progress in identifying new susceptibility genes outside the MHC locus has been slow. With the advent of new global methods for genetic analysis such as large-scale sequencing, gene expression profiling combined with classic linkage analysis and congenic and physical mapping progress is considerably accelerating. Here we review our preliminary work on the use of gene expression mapping to identify new putative genetic pathways contributing to the pathogenesis of EAE.     

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG_35-55 is suspended in complete Freund''s adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.     

Neurovascular damage in experimental allergic encephalomyelitis: a target for pharmacological control

The blood-brain barrier (BBB) is composed of a continuous endothelial layer with pericytes and astrocytes in close proximity to offer homeostatic control to the neurovasculature. The human demyelinating disease multiple sclerosis and the animal counterpart experimental allergic encephalomyelitis (EAE) are characterized by enhanced permeability of the BBB facilitating oedema formation and recruitment of systemically derived inflammatory-type cells into target tissues to mediate eventual myelin loss and neuronal dysfunction. EAE is considered a useful model for examining the pathology which culminates in loss of BBB integrity and the disease is now proving valuable in assessing compounds for efficacy in limiting damage at neurovascular sites. The precise mechanisms culminating in EAE-induced BBB breakdown are unclear although several potentially disruptive mediators have been implicated and have been previously identified as potent effectors of cerebrovascular damage in non-disease related conditions of the central nervous system. The review considers evidence that common mechanisms may mediate cerebrovascular permeability changes irrespective of the initial insult and discusses therapeutic approaches for the control of BBB leakage in the demyelinating diseases.