Central nervous system demyelination and remyelination in multiple sclerosis and viral models of disease

The mechanisms of myelin injury and repair were studied in acute multiple sclerosis lesions and in a murine model of demyelination induced by a virus. Injury to oligodendrocytes resulting in degeneration of inner glial loops and inner myelin lamellae (dying-back oligodendrogliopathy) was observed by electron microscopy in brain biopsies of acute demyelinating lesions. Attempts at central nervous system remyelination as manifested by thinly myelinated axons and proliferation of oligodendrocytes were observed at the edge of many acute plaques. To develop therapeutic strategies to inhibit demyelination or promote remyelination, mice infected intracranially with Theiler's virus (a picornavirus) were studied. Experimental manipulation of Theiler's virus-infected mice by treatment during chronic demyelinating disease with immunoglobulins directed at normal spinal cord antigens or with monoclonal antibodies which deplete CD4 or CD8-positive T cells reslted in augmentation of new myelin synthesis. These observations suggest that disturbances in the myelinating function of oligodendrocytes, events not accompanied by death of these cells, may be among the earliest pathological events in multiple sclerosis. Experiments using the Theiler's virus model of demyelination indicate that manipulation of the immune response has the potential to promote central nervous system remyelination and functional recovery in multiple sclerosis.     

Semliki Forest virus-induced demyelination and remyelination--involvement of B cells and anti-myelin antibodies

Semliki Forest virus (SFV) infection induces a demyelinating encephalomyelitis in the central nervous system (CNS) of mice and serves as a model for multiple sclerosis (MS). This study investigated CNS immune responses at different stages of infection and during SFV-induced demyelination and remyelination. Following the initial CNS inflammation, pathology and viral clearance on days 6-10 post-infection (pi), primary demyelination was observed in cerebellar, brainstem and corpus collosal white matter by days 15-21 pi, with plasma cells and microglia as main participants, and this was followed by remyelination. By day 35 pi, the tissue appeared almost normal. Fluorescent antibody cell sorter (FACS) analysis showed that brain CD8(+) T cells increased during the initial inflammatory response and gradually decreased thereafter. Brain B cell (B220(+)CD19(+)) numbers did not change significantly during the course of infection; however, from days 14 to 35 pi, they matured and produced antibodies to viral and myelin proteins (and peptides) during the period of demyelination and remyelination. The proportion of CD3(-)B220(-)CD11b(+) cells also progressively increased throughout the periods of de- and remyelination. Our results suggest that CD8(+) T cells are involved in the initial destruction of CNS tissue during the first weeks of SFV infection, while B cells, antibodies and microglia may contribute to the myelin pathology seen after recovery.     

Efficient central nervous system remyelination requires T cells

We demonstrate a role for immune functions in the spontaneous remyelination of central nervous system (CNS) axons after lysolecithin-induced demyelination in the spinal cord. Rag-1-deficient mice lack both B cells and T cells and show significantly reduced spontaneous remyelination compared with control mice of matching genetic background. Mice lacking or depleted of either CD4(+) T cells or CD8(+) T cells also exhibit reduced remyelination. These data indicate that T cells are necessary for efficient CNS remyelination. Thus, general nonspecific immunosuppression as a therapeutic approach for the treatment of CNS injury and demyelinating disease may have undesirable effects on subsequent tissue repair.     

Clonal expansions of pathogenic CD8+ effector cells in the CNS of myelin mutant mice

Tissue damage in the CNS is critically influenced by the adaptive immune system. Primary oligodendrocyte damage (by overexpression of PLP) leads to low-grade inflammation of high pathological impact, which is mediated by CD8+ T cells. To yield further insight into pathogenesis and nature of immune responses in myelin mutated mice, we here apply a detailed immunological characterization of CD8+ T cells in PLP-transgenic and aged wild type mice. We provide evidence that T effector cells accumulate in the CNS of PLP-transgenic and wild-type mice and show a higher level of activation in mutant mice, indicated by surface markers and clonal expansions, as demonstrated by T cell receptor CDR3-spectratype analysis. Vbeta-Jbeta similarities suggest specificity against a common antigen, albeit we could not find specific responses against myelin-antigen-derived peptides. The association of primary oligodendrocyte damage with secondary expansions of pathogenic cells underlines the role of adaptive immune reactions in neurodegenerative and neuroinflammatory diseases.     

Transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response

Transplantation of remyelination-competent cells represents a promising strategy for the treatment of demyelinating diseases. As the environment dictates the success or failure of remyelination, it is critical to understand the role that the immune system plays in transplant-mediated remyelination. In this study, we evaluated the severity of neuroinflammation following transplantation of glial-committed progenitor cells into the spinal cords of mice chronically infected with mouse hepatitis virus (MHV), a model in which T cells and macrophages are critical in amplifying the severity of demyelination. Transplantation was performed following viral persistence in which inflammation and demyelination are established and clinical disease is evident. Mice were sacrificed 10 and 21 days following progenitor cell transplantation and the effect on neuroinflammation evaluated. Treatment did not alter accumulation of T cells or macrophages within the CNS as compared to control mice. Moreover, progenitor cell implantation did not affect local cytokine/chemokine gene expression in the CNS. Finally, remyelination associated with transplantation did not result in an imbalance of T(H)1-associated cytokine production by virus-specific T cells. These studies demonstrate that progenitor cell-mediated remyelination is not the result of modulating the composition of the cellular infiltrate nor cytokine expression by virus-specific T cells and suggest that remyelination may not depend on amelioration of the inflammatory response or alteration of cytokine secretion by virus-specific T cells.     

Differential DM20 mRNA expression distinguishes two distinct patterns of spontaneous recovery from murine autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS) mediated by T cells responding to CNS myelin proteins. Immunization of SWXJ mice with the immunodominant p139-151 peptide of myelin proteolipid protein (PLP) results in a relapsing-remitting pattern of EAE characterized by incomplete remyelination during clinical recovery. In the present study we observed two distinct clinical patterns of spontaneous remission during recovery from EAE, viz., sustained remission involving continuous neurologic improvement and aborted remission involving modest transient clinical improvement. We hypothesized that the ability to recover from autoimmune demyelination was directly linked to remyelination events that recapitulated developmental processes. Quantitative immunocytochemistry of CNS tissue showed decreased demyelination in mice undergoing sustained remission compared to mice undergoing aborted remission. Quantitative RT-PCR analysis showed elevated expression of DM20, the developmental isoform of PLP, in CNS tissue from mice undergoing sustained remission compared to mice undergoing aborted recovery. Moreover, DM20 expression was similarly elevated in CNS tissue from mice undergoing sustained recovery from EAE relapse. Our data indicate that expression of the developmental DM20 isoform of PLP is intimately associated with decreased demyelination and sustained clinical recovery from EAE. Thus, DM20 gene expression may provide an appropriate molecular target for promoting CNS remyelination and may serve as a useful marker for predicting clinical outcome and assessing the effectiveness of strategies aimed at promoting CNS tissue repair during autoimmune demyelinating disease.     

Inhibition of Notch signaling enhances tissue repair in an animal model of multiple sclerosis

Oligodendrocytes (OLs) fail to regenerate myelin destroyed by the immune attack in multiple sclerosis (MS) and lesion areas are eventually largely occupied by astrocytic scar tissue. Loss of OLs in MS does not account for the limited myelin repair as lesions contain a considerable number of OL precursor cells (OPC). Activation of the Notch pathway has been shown to provide inhibitory signals for OPC and to hamper their ability to produce myelin during CNS development. Here we show that gamma-secretase inhibition of Notch signaling within OL of CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) significantly enhanced clinical recovery and in the CNS, promoted remyelination and reduced axonal damage. Functional assays confirmed decreased Notch signaling in inhibitor-treated groups. Therefore, gamma-secretase inhibition led to an environment more conducive to myelin repair and axonal survival. Our results suggest that manipulation of the environment associated with Notch activation in the mature CNS provides a promising therapeutic target in MS.     

Human antibodies accelerate the rate of remyelination following lysolecithin-induced demyelination in mice

Immunoglobulin-based therapies are becoming increasingly common for the treatment of neurologic and autoimmune diseases in humans. In this study, we demonstrate that systemic administration of either polyclonal human immunoglobulins or specific human monoclonal antibodies can accelerate the rate of CNS remyelination following toxin-induced demyelination. Injection of lysolecithin directly into the spinal cord results in focal demyelinated lesions. In contrast to other murine models of demyelinating disease, the mechanism of demyelination following lysolecithin injection is independent of immune system activation, and chronic inflammation at the site of the lesion is minimal. Administration of polyclonal human IgM (pHIgM) or a serum-derived human monoclonal antibody (sHIgM22) resulted in approximately a twofold increase in remyelinating axons when compared to animals treated with saline or with antibodies that do not promote repair. Both pHIgM and sHIgM22 show strong binding to CNS white matter and oligodendrocytes, while antibodies that did not accelerate remyelination do not. This differential staining pattern suggests that enhanced remyelination may result from direct stimulation of oligodendrocyte remyelination by binding to surface receptors on oligodendrocytes or glial progenitor cells. We propose the use of human polyclonal IgM or specific human monoclonal IgM antibodies as potential therapies to enhance myelin repair following CNS injury and disease.     

Immunopathogenesis of multiple sclerosis

Multiple sclerosis (MS) is a suspected autoimmune disease in which myelin-specific CD4+ and CD8+ T cells enter the central nervous system (CNS) and initiate an inflammatory response directed against myelin and other components of the CNS. Acute MS exacerbations are believed be the result of active inflammation, and progression of disability is generally believed to reflect accumulation of damage to the CNS, particularly axonal damage. Over the last several years, the pathophysiology of MS is being appreciated to be much more complex, and it appears that the development of the MS plaque involves a large number of cell populations, including CD8+ T lymphocytes, B cells, and Th17 cells (a population of helper T cells that secrete the inflammatory cytokine IL-17). The axonal transection and degeneration that is thought to represent the basis for progressive MS is now recognized to begin early in the disease process and to continue in the progressive forms of the disease. Molecules important for limiting aberrant neural connections in the CNS have been identified, which suppress axonal sprouting and regeneration of transected axons within the CNS. Pathways have also been identified that prevent remyelination of the MS lesion by oligodendrocyte precursors. Novel neuroimaging methodologies and potential biomarkers are being developed to monitor various aspects of the disease process in MS. As we identify the pathways responsible for the clinical phenomena of MS, we will be able to develop new therapeutic strategies for this disabling illness of young adults.     

Self-tolerance in the immune privileged CNS: lessons from the entorhinal cortex lesion model

Upon peripheral immunization with myelin epitopes, susceptible rats and mice develop T cell-mediated demyelination similar to that observed in the human autoimmune disease multiple sclerosis (MS). In the same animals, brain injury does not induce autoimmune encephalomyelitis despite massive release of myelin antigens and early expansion of myelin specific T cells in local lymph nodes, indicating that the self-specific T cell clones are kept under control. Using entorhinal cortex lesion (ECL) to induce axonal degeneration in the hippocampus, we identified possible mechanisms of immune tolerance after brain trauma. Following ECL, astrocytes upregulate the death ligand CD95L, allowing apoptotic elimination of infiltrating activated T cells. Myelin-phagocytosing microglia express MHC-II and the costimulatory molecule CD86, but lack CD80, which is found only on activated antigen presenting cells (APCs). Restimulation of invading T cells by such immature APCs (e.g. CD80 negative microglia) may lead to T cell anergy and/or differentiation of regulatory/Th3-like cells due to insufficient costimulation and presence of high levels of TGF-beta and IL-10 in the CNS. Thus, T cell -apoptosis, -anergy, and -suppression apparently maintain immune tolerance after initial expansion of myelin-specific T lymphocytes following brain injury. This view is supported by a previous metastatistical analysis which rejected the hypothesis that brain trauma is causative of MS (Goddin et al., 1999). However, concomitant trauma-independent proinflammatory signals, e.g., those evoked by clinically quiescent infections, may trigger maturation of APCs, thus shifting a delicate balance from immune tolerance and protective immune responses to destructive autoimmunity.     

Spontaneous remyelination following prolonged inhibition of alpha4 integrin in chronic EAE

Inhibition of alpha(4)beta(1) integrin blocks immune cell influx into the CNS providing benefit to patients with multiple sclerosis and in animal model systems. We have used this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis. We observed (1) 87% of plaques showed remyelination after 40 days of treatment; (2) myelin repair occurred in half of the total lesion area; (3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle-treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitates mechanisms of spontaneous remyelination.     

Remyelination of central nervous system lesions in experimental genital herpes simplex virus infection

To study spinal cord remyelination in a model of genital herpes simplex virus type 2 (HSV-2) infection, adult female mice were inoculated by a vaginal route. At intervals up to 6 months after infection, cord tissues were removed and examined by light and electron microscopy and by immunohistochemical methods. As a consequence of acute infection, 60% of mice developed multifocal central nervous system (CNS) demyelinative lesions in the lower thoracic, lumbar, or upper sacral cord. These lesions, already present 10 days after infection, contained naked axons and mononuclear cells, including macrophages. At 2 weeks, while active myelin breakdown was still ongoing, numerous Schwann cells were present in lesions and surrounded denuded axons. At 3 weeks, the earliest remyelination was seen, and was carried out by Schwann cells and to a lesser extent by oligodendrocytes. Remyelination was extensive by 6-10 weeks and was apparently completed after 3 months. Immunocytochemical studies using antisera to myelin proteins showed relatively distinct zones of central and peripheral remyelination in some lesions, whereas remyelination was of mixed type in others. Thus the remyelinative response following experimental HSV-2-induced CNS demyelination begins promptly, proceeds briskly and goes to completion. With a natural route of inoculation and a relatively avirulent strain of this human pathogen, we have produced a model of CNS white matter injury and repair in a high proportion of infected mice that may be useful in understanding mechanisms of human demyelinative disease.     

Memory cells specific for myelin oligodendrocyte glycoprotein (MOG) govern the transfer of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory disease of the CNS mediated by CD4(+) T cells directed against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin antigens like myelin oligodendrocyte glycoprotein (MOG). We have explored the transfer of EAE using MOG(35-55)-specific TCR transgenic (2D2) T cells. Unsorted 2D2 Th1 cells reliably transferred EAE. Further, we found that CD44(hi)CD62L(lo) effector/memory CD4(+) T cells are likely responsible for the disease transfer due to the up-regulation of CD44. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.     

Stem cells for the treatment of myelin loss

Treatment of myelin loss is particularly suited to therapeutic strategies based on cell replacement. Demyelination represents a defined and functionally debilitating deficit, and remyelination can be accomplished by supplying regions of demyelination with myelinogenic cell populations. Clinical interest in stem cells as a source of myelinogenic cells arises from their ability to provide an apparently unlimited cell supply for transplantation, and from recent demonstrations that they can be directed to myelinogenic phenotypes with high purity. Here, I present the emerging perspective that stem-cell-mediated remyelination of the adult CNS is a viable therapeutic strategy, and discuss the challenges to remyelination posed by the environment of acute and chronic injuries.     

CD8-deficient SJL mice display enhanced susceptibility to Theiler's virus infection and increased demyelinating pathology

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4(+) T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8(+) T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. beta2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8(+) T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4(+) T cells in this capacity and further emphasize the potential for CD8(+) T cells to contribute to protection from TMEV-induced demyelination.     

Overcoming failure to repair demyelination in EAE: gamma-secretase inhibition of Notch signaling

In multiple sclerosis (MS), myelin destroyed by the immune attack is not effectively repaired by oligodendrocytes (OLs) and MS foci eventually undergo glial scarring. Although oligodendrocyte precursor cells (OPCs) are normally recruited to the lesion areas, they fail to mature and remyelinate the damaged fibers. Activation of the Notch pathway has been shown to inhibit OPC differentiation and to hamper their ability to produce myelin during CNS development. We have recently shown that inhibition of gamma-secretase within the CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) blocks Notch pathway activation in OLs, promotes remyelination, reduces axonal damage and significantly enhances clinical recovery from the disease. Our results suggest that inhibiting the non-myelin permissive environment maintained by Notch pathways within the mature CNS offers a new strategy for treating autoimmune demyelination, including MS.     

Pathogenesis of chronic progressive myelopathy associated with human T-cell lymphotropic virus type I

Human T-cell lymphotropic virus type I (HTLV-I) induces a chronic demyelinating disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). While only 0.25% of HTLV-I-infected individuals develop HAM/TSP, the mechanisms responsible for the progression of an HTLV-I carrier state to clinical disease are not clear. In particular, no specific sequence differences have been found between HTLV-I recovered from HAM patients and HTLV-I-infected carriers. Since CD4 T cells are the major reservoir of the virus, at least three hypotheses implicating CD4 T cells directly or indirectly have been proposed: 1) The cytotoxic hypothesis predicts that activated and HTLV-I-infected CD4 T cells migrate to the CNS and infect resident cells. Cytotoxic CD8 T cells may then recognize viral antigens on HTLV-I-infected CNS cells causing a cellularly mediated cytotoxic demyelination. 2) The autoimmune hypothesis predicts that either (a) virally reactive T cells cross-react with a CNS antigen, or (b) random infection of CD4 T cells eventually results in the infection of CNS-autoreactive CD4 T cells that, by virtue of the productive HTLV-I infection, become activated, expand and migrate to the CNS, where they encounter their antigen. This results in a specific immune response and demyelination, as is known to occur in experimental autoimmune encephalomyelitis. 3) The bystander damage hypothesis does not implicate a specific response against CNS cells. Instead this hypothesis suggests that the presence of IFN-γ-secreting HTLV-I-infected CD4 T cells and their recognition by virally specific CD8 T cells in the CNS induce microglia to secrete cytokines, such as TNF-α, which may be toxic for the myelin.     

Spontaneous central nervous system remyelination is not altered in NFH-lacZ transgenic mice after chemical demyelination

Harmonious functioning of the nervous system depends on neuron-glia interactions, particularly between the axons and their myelinating cells, i.e., oligodendrocytes (OL) in the central nervous system (CNS). In human demyelinating diseases such as multiple sclerosis (MS), demyelination may be associated with axonal damage, but alterations of the axonal cytoskeleton, which is composed mainly of neurofilaments (NF) and microtubules, are largely unknown, as are the consequences on remyelination. In a model of demyelination induced by lysophosphatidylcholine (LPC), we have shown that demyelination was correlated with a decrease in NF immunolabelling, and that these axonal abnormalities were reduced by platelet-derived growth factor (PDGF)-enhanced remyelination in adult rats. We have analysed the spontaneous remyelination after LPC stereotaxic injection in the CNS of transgenic NFH-lacZ mice, which present axonal atrophy caused by abnormal distribution of NF, associated with hypermyelination in the PNS, and normal myelin thickness in the CNS. Axonal atrophy in the CNS of NFH-lacZ mice was confirmed, but it was not worsened by demyelination. On the contrary, demyelination induced axonal atrophy in wild-type mice, demonstrating that NF are essential for axonal calibre determination. Moreover, an efficient spontaneous remyelination occurred in NFH-lacZ as well as in wild-type mice, indicating that the NF are not necessary for CNS remyelination. These findings point out that NF modifications observed in MS may not be responsible for the lack of remyelination in this disease.