Studies In Experimental Autoimmune Encephalomyelitis Research Articles

Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-d-aspartate (NMDA) receptor activation

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood–brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO−). Recent studies have implicated ONOO− in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO−. The present investigation examined glutamate-induced ONOO− formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO− production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO− levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO−-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO− production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO− generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.

Tolerance induction by molecular mimicry: prevention and suppression of experimental autoimmune encephalomyelitis with the milk protein butyrophilin.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system. Although the etiology of MS remains unknown, studies in experimental autoimmune encephalomyelitis (EAE) have suggested that foreign molecules, which show molecular mimicry with myelin antigens, may play an important role as causative agents of the human disease. In this study, we investigate the molecular mimicry between the extracellular Ig-like domain of the cow's milk protein butyrophilin (BTN) and the extracellular domain of myelin oligodendrocyte glycoprotein (MOG), a candidate autoantigen in MS. Interestingly, we found that as a result of a non-pathogenic cross-reactivity that is localized to a subdominant region of MOG, treatment of C57BL/6 mice with BTN either before or after immunization with MOG was shown to prevent and also suppress the clinical manifestations of EAE. BTN treatment resulted in a significant reduction in both proliferation and production of Th1-related cytokines (IFN-gamma, IL-2, IL-12 and granulocyte macrophage colony stimulating factor) in response to MOG. This specific inhibition was consistently associated with an up-regulation in IL-10 secretion. Furthermore, adoptive transfer of BTN-specific T cells prior to active immunization with MOG resulted in a transitory reduction of the clinical symptoms. Our results suggest that the clinical improvement associated with BTN treatment involved the combination of both anergy and regulatory cells secreting high levels of IL-10. In conclusion, we show that despite the traditional link between molecular mimicry and pathogenic immune response, environmental agents that share homology with autoantigens may also represent a source of cells with a protective phenotype.

Probable epitopes: Relationships between myelin basic protein antigenic determinants and viral and bacterial proteins.

Nature holds numerous viral and bacterial proteins with regions of similarity to myelin basic protein antigenic determinants. Bioinformatic technology, including sequence similarity searches, may allow for the detection of biochemical and biophysical relationships between these peptides. Understanding these relationships is essential to understanding immune-mediated disease and, consequently, may be used to elucidate the etiology of pathological demyelinating diseases such as multiple sclerosis. Studies of experimental autoimmune encephalomyelitis have been used to identify antigenic determinants. We have used these determinants to search available databases of viral and bacterial proteins. Our results indicate numerous viral and bacterial protein segments with probabilistic sequence similarity to myelin basic protein antigenic determinants.

The role of MCP-1 (CCL2) and CCR2 in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) is the commonest inflammatory demyelinating disease of the human central nervous system (CNS). In MS, CNS inflammation is associated with demyelination and axonal degeneration, which leads to clinical presentation. Expression and cellular localization of CCL2/MCP-1 and CCR2 in MS have been described in the three compartments: brain, cerebrospinal fluid (CSF) and blood. Evidence from descriptive, transgenic, knockout and neutralizing studies of experimental autoimmune encephalomyelitis (EAE) points towards a nonredundant role of CCL2 and CCR2 in the recruitment of inflammatory infiltrate into the CNS. Hence, CCL2 and CCR2 may be targets for specific and effective treatment in MS.

Systemic disease and neuro-ophthalmology: annual update 2000 (Part I).

Multiple sclerosis (MS) is a common demyelinating disease of the central nervous system (1–86), with substantial long-term neurologic consequences (1,4,9,25,34, 52,54,55,57,60,63,65). After 10 years with MS, 50% of patients are unable to perform household and occupational responsibilities; after 15 to 20 years, 50% are unable to walk without assistance; after 25 years, 50% are unable to ambulate. The average annual cost of MS in the United States is greater than 6.8 billion dollars (1). There are three main subtypes of the disease: relapsing remitting (RR), secondary progressive (SP), and primary progressive (PP). This update reviews the current status of MS therapy (1–86). We have chosen to focus on the new and emerging immunomodulatory therapies for disease relapses and the treatments to prevent disease progression. We do not review the treatments for common MS-related sensory and motor symptoms, fatigue, or depression (35).

Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors

Controlled trials in multiple sclerosis (MS) and case reports in acute demyelinating encephalomyelitis (ADEM) have shown that intravenous immunoglobulins (IVIg) are of therapeutic benefit in central nervous system (CNS) inflammatory diseases. Studies in experimental autoimmune encephalomyelitis (EAE) have suggested these effects are mediated by modulation of the cytokine network and T cell responses. However, there are no data on the influence of IVIg on the local immune reaction in the CNS, the site of inflammation in EAE. We have therefore studied the effect of IVIg on cultured rat microglia, the main immune cell in the CNS. IVIg increased nitric oxide (NO) production in a dose-dependent manner in microglia stimulated with IFNγ. The increase was only marginal in LPS-treated cells, and no effect was seen in untreated microglia or after stimulation with TNFα or PMA. This enhancement of NO production depended on the Fc portion of IVIg and could be abrogated by the pharmacological inhibition of Syk and phosphatidylinositol 3-kinase, two enzymes involved in the signalling cascade of Fc receptors. TNFα secretion was dose-dependently stimulated by IVIg in both untreated microglia and after stimulation with LPS or IFNγ. Again, this effect was mediated through the Fc portion. Finally, we showed that Fc receptor-mediated phagocytosis was inhibited by IVIg, presumably by blockade of the Fc receptor. These different effects may protect oligodendrocytes from antibody mediated phagocytosis and on the other hand could terminate the immune reaction by induction of apoptosis in infiltrating T cells via NO and TNFα. We propose that IVIg, in addition to known effects on the peripheral immune system, may also modulate the local immune reaction in CNS inflammatory disease.

Mechanisms of central and peripheral T-cell tolerance: lessons from experimental models of multiple sclerosis.

Studies of experimental autoimmune encephalomyelitis in conventional and transgenic mouse models have clarified mechanisms of central and peripheral tolerance to myelin antigens. It is now clear that myelin antigens are expressed in the thymus and their expression can influence generation of the potential autoimmune T-cell repertoire. How autoreactive T cells escape tolerance in the thymus is largely unclear. One mechanism has been revealed through the use of a transgenic mouse expressing a T-cell receptor specific for a myelin antigen. T cells specific for the N-terminal epitope of myelin basic protein escape tolerance through low avidity interaction. The affinity of antigen binding to MHC also proves to be important for induction of peripheral tolerance. Peptides may be administered in solution to adults in order to reinstate peripheral tolerance and suppress disease. Induction of antigen-specific suppression with synthetic peptides can result in either linked or bystander suppression and this appears to involve the generation of T cells secreting suppressive cytokines. The use of altered peptide ligands for induction of peripheral tolerance has been investigated. This can be achieved but the complexity of the approach argues against its use for treatment of human autoimmune diseases.

Apoptosis in brain-specific autoimmune disease

Recent neuropathological studies of experimental autoimmune encephalomyelitis have focused attention on the high number of cells in the lesions that show typical morphological features of apoptosis. Surprisingly, it has turned out that the vast majority of apoptotic cells are T lymphocytes and that they actually represent the antigen-specific T-cell population responsible for the induction of the disease. Taken together, these data suggest that clearance of autoimmune inflammation in the nervous system is accomplished by the destruction of the antigen-specific T-cell population within the lesions. This may explain the low level of central nervous system specific T-cell memory formation, as well as previously unexplained phenomena of ‘epitope spreading’, in autoimmune inflammation of the nervous system

A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats.

We compared the T cell responses of Lewis (LEW) and DA rats to guinea pig myelin basic protein (MBP), the synthetic peptides corresponding to the epitopes that are encephalitogenic in the LEW strain (MBP73-86, MBP68-86, and MBP87-99), and bovine proteolipid protein (PLP). DA and LEW rats were susceptible to experimental autoimmune encephalomyelitis (EAE) induced with MBP or MBP68-86, but the peptide was less active in DA rats than in intact MBP molecule. MBP73-86 and MBP87-99 induced EAE in LEW rats but not in the DA strain. MBP89-169 was also encephalitogenic in DA rats. Encephalitogenic CDa+ T cell lines and clones derived from MBP-sensitized DA rats secreted IFN-gamma and TNF-alpha and proliferated to MBP and MBP89-169, but not to MBP68-88. However, T cells from MBP68-86-sensitized DA or LEW rats proliferated specifically in an I-A-restricted response to MBP68-86. T cells from MBP87-99-immunized LEW rats responded to MBP87-99 in the context of I-E, whereas the peptide-specific response of MBP87-99 immunized DA rats was I-A-restricted, although FACScan analysis indicated that DA rats express both I-A and I-E. DA rats were also highly susceptible to EAE induced with PLP; 0.6 nmol was Encephalitogenic for DA rats, but did not induce clinical EAE in LEW rats. Although both DA and LEW rats are highly susceptible to EAE, we demonstrate marked differences in the array of myelin epitopes capable of inducing the disease, as well as the MHC restriction of these epitopes, between the two rat strains.

The clonal composition of myelin basic protein-reactive encephalitogenic T cell populations is influenced both by the structure of relevant antigens and the nature of antigen-presenting cells.

Studies of experimental autoimmune encephalomyelitis (EAE) in rodents have revealed that encephalitogenic T cell lines reactive with myelin basic protein (BP) are frequently dominated by clones expressing a restricted T cell receptor repertoire. Using the rat EAE model, we have begun to examine the basis for clonal dominance within BP-reactive T cell lines. We find that variations introduced into the standard protocol of periodic antigen stimulation produce marked shifts in the representation of different clones within encephalitogenic T cell populations. For example, altering the source of antigen-presenting cells (APC), while holding antigen (BP) constant, and substituting BP from guinea pig (GPBP) for that of the rat antigen (RBP) with constant APC, both cause shifts in the composition of the dominant clones within BP-reactive T cell lines. Our results suggest that: (i) adherence to an invariant protocol of antigen challenge may lead to an underestimation of the diversity of BP-reactive encephalitogenic T cell populations; and (ii) the minor structural differences between GPBP and RBP not only cause the weak immunogenicity of RBP but also result in the alteration of different T cell subsets. These observations indicate that apparent restrictions upon the repertoire of autoimmune T cells should be interpreted with caution when such cells are elicited by immunization with foreign antigens.

Evidence for genetic contamination of inbred Buffalo rats (RT-1b) obtained from a commercial vendor

Inbred Buffalo rats (RT-1b) have been used in studies of experimental autoimmune encephalomyelitis and autoimmune thyroiditis. Since our studies, and those of others, have relied on the genetic purity of inbred Buffalo rats, we chose to test these animals for expression of strain-dependent, allotype-specific variants of CD45 (leukocyte common antigen, LCA) using the monoclonal antibodies RT7.1 and RT7.2. The goal of this study was to confirm the genetic purity and to verify the inbred status of Buffalo rats obtained from a commercial source.

Restricted T-cell receptor V beta gene usage by myelin basic protein-specific T-cell clones in multiple sclerosis: predominant genes vary in individuals.

Recent studies in experimental autoimmune encephalomyelitis as a model for multiple sclerosis (MS) have demonstrated limited heterogeneity in T-cell antigen receptors (TCR) specific for myelin basic protein (MBP). To investigate restricted beta-chain variable-region (V beta) gene usage in humans, we analyzed TCR gene rearrangements in two lines and 34 MBP-specific T-cell clones that were isolated from five MS patients and two healthy subjects. The T cells were characterized for their specificity to MBP epitopes and HLA-restricting molecules. We demonstrate here that MBP-specific T-cell clones from these different MS patients and healthy individuals, in contrast to T cells from rodents, display a more diverse V beta gene usage as evidenced by their TCR V beta gene rearrangements. However, the different MBP-specific T-cell clones isolated from each individual MS patient showed a common V beta gene usage, suggesting individual-specific TCR restriction. Out of 16 MBP-specific clones derived from a single MS patient, 12 clones (75%) utilized the V beta 15 gene for their TCR gene rearrangement. MBP-specific clones isolated from four other MS patients also showed a consistent tendency for a predominant, but different, TCR V beta gene rearrangement. These results suggest a TCR heterogeneity among MBP-specific T-cell clones from different individuals but a limited TCR V beta gene usage among MBP-specific T-cell clones of the same individual. The predominant V beta gene used by the MBP-specific T-cell clones studied here was not found to correlate with the epitope specificity of T cells or with their restricting HLA molecule. These findings may support the possibility of intervention with monoclonal antibodies to specific V beta gene products as an approach to immune therapy of MS but also imply the necessity for an individual-specific immunotherapeutic approach.

An immunohistological study of autoimmune encephalomyelitis and neuritis in the rabbit: Observations in the dorsal root ganglion using the freeze-dried paraffin-embedded tissue technique

Previous studies of experimental autoimmune encephalomyelitis have shown that, in the central nervous system, the emigration of T-lymphocytes precedes that of mononuclear phagocytes during inflammatory lesion formation. In the present report, the formation of analogous lesions of autoimmune neuritis (EAN) was investigated in the dorsal root ganglia of rabbits immunized with homologous spinal cord in Freund's adjuvant. The relative time course of emigration of T-lymphocytes and mononuclear phagocytes into the ganglia was examined using monoclonal antibody labeling of both types of cells in serial sections of freeze-dried paraffin-embedded tissue. Results indicate that, unlike in the central nervous system, in the rabbit dorsal root ganglion T-lymphocytes and mononuclear phagocytes appear to emigrate simultaneously, as revealed by their concomitant presence in the earliest detectable lesions of EAN. It was also found that the cortical region of the rabbit dorsal root ganglion was a preferential site of EAN lesion formation, and that such lesions correlated well with the onset of clinical signs of paralysis. These results are discussed within the context of known “blood-tissue barriers” and the possible local modulation of inflammatory cell entry into regions of the nervous system.

Simultaneous visualization of vascular permeability change and leukocyte egress in the central nervous system during autoimmune encephalomyelitis.

An unresolved issue in the study of demyelinating disease is whether blood-brain barrier damage is dependent upon the migration of inflammatory cells into the central nervous system (CNS). In a study of experimental autoimmune encephalomyelitis (EAE) in rabbits, a freeze-dried, paraffin-embedded tissue technique was exploited to enable (1) the immobilization of intravenously injected sodium fluorescein tracer, as an index of vascular permeability; and (2) an effective labeling by monoclonal antibodies of both T-lymphocytes and mononuclear phagocytes in "unfixed" neural tissue. Using these newly combined methods, evidence was found that increased vascular permeability in the CNS during EAE occurs concomitantly with, and not prior to, infiltration by mononuclear phagocytes.

IMMUNOPHARMACOLOGICAL DISEASE: A BREAK IN TOLERANCE TO RECEPTOR SITES

This hypothesis invokes a new pathophysiological process to explain functional lesions in certain organ-specific autoimmune diseases. It is suggested that a pathogenic immune response can be directed against exposed cell-membrane receptor sites for humoral agents such as neurotransmitters or peptide hormones, and that interaction of an antibody or immunocyte with the receptor site causes an " immunopharmacological" block which disrupts the normal function of that cell and related cells. Tissue damage would result either from the ensuing inflammatory response, initiated by the immunological interaction with the tissue receptor(s), or from disuse atrophy of the unstimulated target cells. This hypothesis was developed from studies in experimental autoimmune encephalomyelitis (E.A.E.) and could be applicable to human multiple sclerosis. The autoantigen responsible for inducing E.A.E. is the small" tryptophan peptide" isolated from the basic protein of myelin, and apparently present also in membranes of nerve-endings. The three-dimensional structure of the tryptophan peptide fulfils the calculated requirement for a central-nervous-system receptor site for 5-hydroxytryptamine (5-H.T.). Antibody to the peptide would compete with 5-H.T. for the receptor site. The concept of functional failure due to immuno-pharmacological block is applicable to other diseases with abnormal immunological features including myasthenia gravis, with its characteristic motor end-plate lesion, and autoimmune diseases of secretory organs. The postulated functionally disruptive autoantibodies may be detectable better by physiological test systems than by conventional immunological tests.