Glutamate excitotoxicity in a model of multiple sclerosis.

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.

Identification of autoantibodies associated with myelin damage in multiple sclerosis.

The molecular mechanisms underlying myelin sheath destruction in multiple sclerosis lesions remain unresolved. With immunogold-labeled peptides of myelin antigens and high-resolution microscopy, techniques that can detect antigen-specific antibodies in situ, we have identified autoantibodies specific for the central nervous system myelin antigen myelin/oligodendrocyte glycoprotein. These autoantibodies were specifically bound to disintegrating myelin around axons in lesions of acute multiple sclerosis and the marmoset model of allergic encephalomyelitis. These findings represent direct evidence that autoantibodies against a specific myelin protein mediate target membrane damage in central nervous system demyelinating disease.

Upregulation and coexpression of adhesion molecules correlate with relapsing autoimmune demyelination in the central nervous system.

The expression of adhesion molecules on central nervous system (CNS) vessels was examined during chronic relapsing experimental autoimmune encephalomyelitis in the SJL mouse. Two molecules associated with cell adhesion were studied: MECA-325, a murine lymph node high endothelial venule marker; and MALA-2, the murine homologue of intercellular adhesion molecule 1. During initial disease, upregulated coexpression of these two molecules occurred in the CNS. This correlated with inflammatory cell invasion. During remission, expression was downregulated, and each subsequent relapse was accompanied by corresponding upregulation. Thus, up- and downregulation of adhesion molecules in the target organ appeared to form an integral part of the inflammatory process in this autoimmune condition and support a role for receptor-mediated inflammatory cell invasion of relevance to the pathogenesis of multiple sclerosis.

Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease.

The properties and outcome of an immune response are best predicted by the lymphokine phenotype of the responding T cells. Cytokines produced by CD4+ T helper type 1 (Th1) T cells mediate delayed type hypersensitivity (DTH) and inflammatory responses, whereas cytokines produced by Th2 T cells mediate helper T cell functions for antibody production. To determine whether induction of Th2-like cells would modulate an inflammatory response, interleukin 4 (IL-4) was administered to animals with experimental allergic encephalomyelitis (EAE), a prototypic autoimmune disease produced by Th1-like T cells specific for myelin basic protein (MBP). IL-4 treatment resulted in amelioration of clinical disease, the induction of MBP-specific Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines in the central nervous system (CNS). Modulation of an immune response from one dominated by excessive activity of Th1-like T cells to one dominated by the protective cytokines produced by Th2-like T cells may have applicability to the therapy of certain human autoimmune diseases.

Myelin basic protein-specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather than protect them from the disease.

Chronic inflammatory autoimmune diseases such as multiple sclerosis, diabetes, and rheumatoid arthritis are caused by CD4(+) Th1 cells. Because Th2 cells antagonize Th1 cell functions in several ways, it is believed that immune deviation towards Th2 can prevent or cure autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease used as a model for multiple sclerosis. Using an adoptive transfer system we assessed the role of Th1 and Th2 cells in EAE. In vitro generated Th1 and Th2 cells from myelin basic protein (MBP)-specific TCR transgenic mice were transferred into normal and immunodeficient mice. Th1 cells caused EAE in all recipients after a brief preclinical phase. Surprisingly, Th2 cells also caused EAE in RAG-1 KO mice and in alphabeta T cell-deficient mice, albeit after a longer preclinical phase. Normal or gammadelta T cell-deficient mice were resistant to EAE induced by Th2 cells. The histopathological features of this disease resembled those of an allergic process. In addition, disease induction by Th1 cells was not altered by coadmininstration of Th2 cells in any of the recipients. These findings indicate that MBP-specific Th2 cells have the potential to induce EAE and that the disease induced by previously activated Th1 cells cannot be prevented by normal lymphocytes nor by previously activated Th2 cells.

Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

Effects of thallium salts on neuronal mitochondria in organotypic cord-ganglia-muscle combination cultures.

A functionally coupled organotypic complex of cultured dorsal root ganglia, spinal cord peripheral nerve, and muscle has been employed in an experimental approach to the investigation of the neurotoxic effects of thallium. Selected cultures, grown for up to 12 wk in vitro, were exposed to thallous salts for periods ranging up to 4 days. Cytopathic effects were first detected after 2 h of exposure with the appearance of considerably enlarged mitochondria in axons of peripheral nerve fibers. With time, the matrix space of these mitochondria became progressively swollen, transforming the organelle into an axonal vacuole bounded by the original outer mitochondrial membrane. Coalescence of adjacent axonal vacuoles produced massive internal axon compartments, the membranes of which were shown by electron microprobe mass spectrometry to have an affinity for thallium. Other axoplasmic components were displaced within a distended but intact axolemma. The resultant fiber swelling caused myelin retraction from nodes of Ranvier but no degeneration. Impulses could still propagate along the nerve fibers throughout the time course of the experiment. Comparable, but less severe changes were seen in dorsal root ganglion neurons and in central nerve fibers. Other cell types showed no mitochondrial change. It is uncertain how these findings relate to the neurotoxic effects of thallium in vivo, but a sensitivity of the nerve cell and especially its axon to thallous salts is indicated.

Suppression of chronic allergic encephalomyelitis: relevance to multiple sclerosis.

The expression of chronic relapsing experimental allergic encephalomyelitis in strain 13 guinea pigs was suppressed with a single series of injections of myelin basic protein in incomplete Freund's adjuvant. The suppression appeared permanent, and subsequent rechallenge with central nervous system antigen failed to elicit exacerbations.

Multiple sclerosis: distribution of T cell subsets within active chronic lesions.

The distribution of T cells and T cell subsets was examined within the human central nervous system in active lesions from seven patients with chronic multiple sclerosis. The monoclonal antibodies anti-T11, anti-T4, and anti-T8 were used to detect total (whole) T cells, helper T cells, and suppressor-cytotoxic T cells, respectively, and a monoclonal antibody against human Ia was used for macrophages and B cells. Lesion progression was associated with large numbers of T4+ cells at the lesion margin and these extended great distances into the adjacent normal-appearing white matter. T8+ cells were most commonly concentrated around the lesion margin and displayed a preferential perivascular distribution. Within the lesion center, only a few T cells were found. Ia+ macrophages were most numerous within the centers of active lesions and were always present in the adjacent normal white matter. The monoclonal antibodies to T cells did not cross-react with glial cells including oligodendrocytes. These results indicate that T4+ cells are actively involved in lesion extension and Ia+ cells, in demyelination.

Axons: isolation from mammalian central nervous system.

Centrifugation of a homogenate of white matter, in a solution of buffered sucrose containing salt, produces a floating layer of myelinated axons. When these are suspended in hypotonic buffer, the mnyelin swells and strips away from the axon. Axons are then separated from the myelin by centrifugation. The resulting preparation consists of a variable population of processes with lengths up to 200 micrometers and diameters between 0.3 and 5.0 micrometers. The axons contain neurofilaments and mitochondria, although no axolemma or neurotubules are evident. The preparation contains cerebroside and sulfatide, yet is essentially free of myelin.

Autoimmune encephalomyelitis: simultaneous identification of T and B cells in the target organ.

Monoclonal antibodies to guinea pig T cells and antibodies to guinea pig immunoglobulin G were used in immunofluorescence studies to identify T and B cells in central nervous system tissue from guinea pigs with acute autoimmune encephalomyelitis. T cells appeared before B cells and were distributed within the white matter parenchyma, while B cells remained in perivascular spaces.

Measles virus matrix protein synthesized in a subacute sclerosing panencephalitis cell line.

Measles virus generally produces acute illness. Rarely, however, persistent infection of brain cells occurs, resulting in a chronic and fatal neurological disease, subacute sclerosing panencephalitis (SSPE). Evidence indicates that expression of the measles virus matrix protein is selectively restricted in this persistent infection, but the mechanism underlying this restriction has not been identified. Defective translation of matrix messenger RNA has been described in one SSPE cell line. This report presents evidence that in a different SSPE tissue culture cell line IP-3-Ca, the matrix protein is synthesized but fails to accumulate. A general scheme is proposed to reconcile the different levels at which restriction of matrix protein has been observed.

T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis.

Encounters with antigen can stimulate T cells to become activated and proliferate, become nonresponsive to antigen, or to die. T cell death was shown to be a physiological response to interleukin-2-stimulated cell cycling and T cell receptor reengagement at high antigen doses. This feedback regulatory mechanism attenuates the immune response by deleting a portion of newly dividing, antigen-reactive T cells. This mechanism deleted autoreactive T cells and abrogated the clinical and pathological signs of autoimmune encephalomyelitis in mice after repetitive administration of myelin basic protein.

Late complications of immune deviation therapy in a nonhuman primate.

The administration of antigens in soluble form can induce antigen-specific immune tolerance and suppress experimental autoimmune diseases. In a marmoset model of multiple sclerosis induced by myelin oligodendrocyte glycoprotein (MOG), marmosets tolerized to MOG were protected against acute disease, but after tolerization treatment a lethal demyelinating disorder emerged. In these animals, MOG-specific T cell proliferative responses were transiently suppressed, cytokine production was shifted from a T helper type 1 (TH1) to a TH2 pattern, and titers of autoantibodies to MOG were enhanced. Thus, immune deviation can increase concentrations of pathogenic autoantibodies and in some circumstances exacerbate autoimmune disease.

GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination.

To investigate the structural role of glial fibrillary acidic protein (GFAP) in vivo, mice carrying a null mutation in GFAP were generated. In 7/14 mutant animals older than 18 months of age, hydrocephalus associated with white matter loss was detected. Mutant mice displayed abnormal myelination including the presence of actively myelinating oligodendrocytes in adults, nonmyelinated axons in optic nerve, and reduced myelin thickness in spinal cord. White matter was poorly vascularized and the blood-brain barrier was structurally and functionally impaired. Astrocytic structure and function were abnormal, consisting of shortened astrocytic cell processes, decreased septation of white matter, and increased CNS extracellular space. Thus, GFAP expression is essential for normal white matter architecture and blood-brain barrier integrity, and its absence leads to late-onset CNS dysmyelination.

Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions.

Multiple sclerosis (MS) brain tissue, spleen, and PBMC were studied using immunocytochemistry and FACS for immunoreactivity for lymphotoxin (LT) and TNF. Both cytokines were identified in acute and chronic active MS lesions but were absent from chronic silent lesions. LT was associated with CD3+ lymphocytes and Leu-M5+ microglia cells at the lesion edge and to a lesser extent, in adjacent white matter. TNF was associated with astrocytes in all areas of the lesion, and with foamy macrophages in the center of the active lesion. In acute lesions, immunoreactivity for TNF in endothelial cells was noted at the lesion edge. No LT or TNF reactivity was detected in Alzheimer's or Parkinson's disease brain tissues but was present at lower levels in central nervous system (CNS) tissue from other inflammatory conditions, except for adrenoleucodystrophy which displayed high levels of LT in microglia. No increase in LT and TNF reactivity was detected in spleen and PBMC of MS patients suggesting specific reactivity within the CNS. These results indicate that LT and TNF may be involved in the immunopathogenesis of MS, and can be detected in both inflammatory cells and cells endogenous to the CNS.

Multiple sclerosis: immune system molecule expression in the central nervous system.

The fundamental message emerging from immunologic and immunopathologic analyses of the brain and spinal cord in multiple sclerosis (MS) is that during inflammation, the central nervous system (CNS) is capable of interactions with the lymphoid system, mainly through induced (as opposed to constitutive) expression of immune system-specific molecules on CNS elements. CNS endothelium, astrocytes and microglial cells are the main participants, with oligodendrocytes and neurons remaining essentially inert. There appears to be nothing unique about the manner in which the CNS responds to inflammation or in the molecules expressed. The ensuing adhesion molecules, pro-inflammatory and regulatory cytokines, histocompatibility molecules, and T and B cell markers, are difficult to distinguish from those occurring in peripheral lymphoid tissue. However, differences certainly exist in the outcome of an inflammatory insult in the CNS versus other, peripheral tissues, whereby there is generally a poor reparatory response. Reasons for the latter appear to lie in the anatomical complexity of the CNS, its vulnerability to damage by soluble mediators, and in the white matter (the battlefield for the inflammatory attack in MS), the exquisite sensitivity of the oligodendrocyte and its myelin to exogenous factors. With the aid of examples drawn from experimental allergic encephalomyelitis, the prime animal model for MS, a number of approaches to prevent or downregulate CNS inflammation during immune-mediated demyelination are presented as possible therapeutic avenues for MS, some of which are already under investigation.

Multiple sclerosis: remyelination in acute lesions.

Central nervous system (CNS) remyelination has been found by light and electron microscopy and immunocytochemistry within fresh lesions from four cases of acute and chronic progressive multiple sclerosis (MS). The remyelination was sparse and existed in an edematous parenchyma containing oligodendrocytes, macrophages, hypertrophic astrocytes and inflammatory components, but no fibrillary astrogliosis. It is suggested that this type of CNS remyelination in MS is a transient event. Possible underlying factors are discussed.

SP-40,40 immunoreactivity in inflammatory CNS lesions displaying astrocyte/oligodendrocyte interactions.

Immunoreactivity for SP-40,40, a putative complement inhibitor, adhesion or protective molecule, has been examined in a variety of inflammatory CNS lesions that displayed associations between hypertrophic astrocytes and oligodendrocytes, a phenomenon previously suggested to be related to oligodendrocyte phagocytosis or protection. SP-40,40 staining was common and was predominantly limited to hypertrophic astrocytes within lesion areas and diminished beyond the lesion margin. However, there was no consistent relationship between SP-40,40 immunoreactivity and astrocytes associated with oligodendrocytes. Staining for terminal complement complex (C5b-9/SC5b-9) occurred in association with larger vessel walls and microglial cells in the most active lesions, but was never seen in hypertrophic astrocytes. No association between SP-40,40 and complement deposition could be demonstrated. Staining for tumor necrosis factor-alpha showed a few scattered hypertrophic astrocytes to be positive. The findings confirm the presence of these astrocyte/oligodendrocyte interactions in active CNS lesions of varied etiology (multiple sclerosis, stroke and AIDS encephalitis). SP-40,40 immunoreactivity was common to hypertrophic astrocytes regardless of their associations with oligodendrocytes but showed no colocalization with terminal complement complex. Thus, these glial interactions do not apparently involve protection against complement-mediated lysis. Furthermore, the presence of SP-40,40 in astrocytes lacking association with oligodendrocytes did not support a role for this protein functioning as an adhesion molecule in astrocyte/oligodendrocyte associations.