The effect of apoptosis inhibitors on experimental autoimmune encephalomyelitis: apoptosis as a regulatory factor

The effect of apoptosis inhibitors on experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, was investigated by intraperitoneal or intracisternal administration of apoptosis inhibitors Ac-YVAD-cmk and zVAD-fmk. After onset of the disease, these agents had no suppressive effect on EAE and resulted in impaired recovery or earlier relapse. Histological examination revealed that administration of zVAD-fmk suppressed the apoptotic death of inflammatory cells in the central nervous system (CNS) of mice with EAE. The results indicated that the apoptotic elimination of infiltrated cells in the CNS might be one of the recovery mechanisms in EAE.     

Effect of diphenyl diselenide on the development of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory and demyelinating disease of the central nervous system with clinical and pathological similarities with multiple sclerosis. The oxidative stress is one of the major mediators of demyelination and axonal damage in both, multiple sclerosis and EAE. Therefore, several studies are being performed to assess whether treatment with antioxidants prevents the progression of these diseases. Some organic forms of selenium that exhibit glutathione peroxidase-like activity have become good candidates for disease prevention and therapy since they catalytically remove oxidative stressors. Particularly, diphenyl diselenide ((PhSe)₂) exerts antioxidant activity and has neuroprotective effects in several systems. The aim of the present study was to prove the therapeutic activity of (PhSe)₂ on the development of EAE. Intraperitoneally administered (PhSe)₂ (1–25 μmoles/kg body weight/day) reduced the incidence of the disease but was also deleterious for the animals. Conversely, (PhSe)₂ given orally (80 μmoles/kg body weight/day) produced a significant inhibition of EAE without any toxic effect. In addition, there was a reduction of the characteristic histological alterations and a diminished in vivo and in vitro T-cell response against the encephalitogenic myelin basic protein. These results show an effective suppression of the autoimmune response that could be the base for future developments of successful antioxidants therapies in EAE as well as in multiple sclerosis.     

Effect of diphenyl diselenide on the development of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory and demyelinating disease of the central nervous system with clinical and pathological similarities with multiple sclerosis. The oxidative stress is one of the major mediators of demyelination and axonal damage in both, multiple sclerosis and EAE. Therefore, several studies are being performed to assess whether treatment with antioxidants prevents the progression of these diseases. Some organic forms of selenium that exhibit glutathione peroxidase-like activity have become good candidates for disease prevention and therapy since they catalytically remove oxidative stressors. Particularly, diphenyl diselenide ((PhSe)₂) exerts antioxidant activity and has neuroprotective effects in several systems. The aim of the present study was to prove the therapeutic activity of (PhSe)₂ on the development of EAE. Intraperitoneally administered (PhSe)₂ (1-25 μmoles/kg body weight/day) reduced the incidence of the disease but was also deleterious for the animals. Conversely, (PhSe)₂ given orally (80 μmoles/kg body weight/day) produced a significant inhibition of EAE without any toxic effect. In addition, there was a reduction of the characteristic histological alterations and a diminished in vivo and in vitro T-cell response against the encephalitogenic myelin basic protein. These results show an effective suppression of the autoimmune response that could be the base for future developments of successful antioxidants therapies in EAE as well as in multiple sclerosis.     

Coronin 1-mediated naive T cell survival is essential for the development of autoimmune encephalomyelitis

Autoimmune encephalomyelitis is a disease of the CNS that can develop when an initial peripheral inflammatory stimulus is followed by infiltration and reactivation of T lymphocytes in the CNS. We report a crucial role for coronin 1, which is essential for maintenance of the naive T cell pool, for the development of murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In the absence of coronin 1, immunization with myelin oligoglycoprotein (MOG(35-55)) peptide largely failed to induce EAE symptoms, despite normal mobilization of leukocyte subsets in the blood, as well as effector cytokine expression comparable with wild-type T cells on polyclonal stimulation. Susceptibility of coronin 1-deficient mice to EAE induction was restored by transfer of wild-type CD4(+) T cells, suggesting that the observed resistance of coronin 1-deficient mice to EAE development is T cell intrinsic. Importantly, although coronin 1-deficient regulatory T cells (Tregs) showed a suppressor activity comparable with wild-type Tregs, Treg depletion failed to restore EAE development in coronin 1-deficient animals. These results suggest a hitherto unrecognized role of naive T cells in the development of autoimmune encephalomyelitis and reveal coronin 1 as a crucial modulator of EAE induction.     

A combined NMR and molecular dynamics simulation study to determine the conformational properties of agonists and antagonists against experimental autoimmune encephalomyelitis

Myelin basic protein (MBP) is one of the best characterized autoantigens causing multiple sclerosis (MS), via a procedure that involves a stable formation of the trimolecular complex of a T-cell Receptor (TCR), an MBP epitope, and the receptor HLA-DR2b. Experimental autoimmune encephalomyelitis (EAE) is considered as an instructive model for MS in humans, and plenty of X-ray data is available for a number of EAE inducing peptide-receptor complexes. To date, though, there are no data available for complexes involving peptides reversing EAE, namely antagonists. Conformational properties of the EAE inducing epitope MBP(87-99) were analyzed in DMSO using the NOE connectivities and vicinal H(N)-H(alpha) coupling constants, and compared with the antagonist altered peptide ligands. A robust method, which is based on a combination of molecular dynamics and energy minimization, is proposed for identifying the putative bioactive conformations. Generated conformations are compared with the known X-ray structure of MBP(83-96) (human sequence numbering) in the HLA-DR2b complex. The structural motif for the agonist-antagonist activity is discussed.     

2-BFI attenuates experimental autoimmune encephalomyelitis-induced spinal cord injury with enhanced B-CK, CaATPase, but reduced calpain activity

The lack of disease-modifying pharmacological agents for effective treatment of multiple sclerosis (MS) still represents a large and urgent unmet medical need. Our previous studies showed that ligands to type 2 imidazoline receptors (I₂R) were effective in protecting spinal cord injury caused by experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this study, we further examined the protective property of a very selective ligand of I₂R, 2-(2-benzofuranyl) 2-imidazoline (2-BFI) against EAE. Importantly, a mechanism of 2-BFI-mediated protection was investigated which possibly involves an I₂R binding protein, brain-creatine kinase (B-CK), as well as CaATPase and calpain. The enzymatic activity of B-CK and CaATPase was significantly reduced in EAE injured spinal cord. Reduction of B-CK activity in EAE spinal cord may lead to energy reduction and dysfunction in cellular calcium homeostasis. Increased intracellular calcium evokes elevation of calpain activity occurring in EAE spinal cord which causes further tissue damage. Indeed, EAE injured spinal cord showed significant reduction in CaATPase and increase calpain activities. Remarkably, spinal cord tissue from mice treated daily with 2-BFI during the progression of EAE significantly restored B-CK and CaATPase enzymatic activities and showed no induction in calpain activity. Moreover, EAE spinal cord from 2-BFI treated mice also demonstrated better preservation of myelin; reduced axonal injury, as evidenced by the lower level of β-APP expression, and above all, highly improved neurobehavioral scores (p < 0.01; n = 10). These findings suggest that 2-BFI can be further developed as a therapeutic drug for MS treatment.     

The CB(2) cannabinoid receptor controls myeloid progenitor trafficking: involvement in the pathogenesis of an animal model of multiple sclerosis

Cannabinoids are potential agents for the development of therapeutic strategies against multiple sclerosis. Here we analyzed the role of the peripheral CB(2) cannabinoid receptor in the control of myeloid progenitor cell trafficking toward the inflamed spinal cord and their contribution to microglial activation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE). CB(2) receptor knock-out mice showed an exacerbated clinical score of the disease when compared with their wild-type littermates, and this occurred in concert with extended axonal loss, T-lymphocyte (CD4(+)) infiltration, and microglial (CD11b(+)) activation. Immature bone marrow-derived CD34(+) myeloid progenitor cells, which play a role in neuroinflammatory pathologies, were shown to express CB(2) receptors and to be abundantly recruited toward the spinal cords of CB(2) knock-out EAE mice. Bone marrow-derived cell transfer experiments further evidenced the increased contribution of these cells to microglial replenishment in the spinal cords of CB(2)-deficient animals. In line with these observations, selective pharmacological CB(2) activation markedly reduced EAE symptoms, axonal loss, and microglial activation. CB(2) receptor manipulation altered the expression pattern of different chemokines (CCL2, CCL3, CCL5) and their receptors (CCR1, CCR2), thus providing a mechanistic explanation for its role in myeloid progenitor recruitment during neuroinflammation. These findings demonstrate the protective role of CB(2) receptors in EAE pathology; provide evidence for a new site of CB(2) receptor action, namely the targeting of myeloid progenitor trafficking and its contribution to microglial activation; and support the potential use of non-psychoactive CB(2) agonists in therapeutic strategies for multiple sclerosis and other neuroinflammatory disorders.     

Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.     

A transgenic model of central nervous system autoimmunity mediated by CD4+ and CD8+ T and B cells

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.     

DM-20, a proteolipid apoprotein, is an encephalitogen of acute and relapsing autoimmune encephalomyelitis in mice

Experimental allergic encephalomyelitis (EAE) was successfully induced in BALB/c mice with DM-20, a protein component of proteolipid apoprotein. DM-20 was separated by ion exchange column chromatography with CM-Trisacryl from proteolipid apoprotein obtained from bovine spinal cords. Its purity was ascertained by SDS-polyacrylamide gel electrophoresis, a dot immunobinding procedure, and amino acid analysis. Nine of 15 animals with a single injection of 100 micrograms of DM-20 and five of seven animals with a booster injection developed hind leg paralysis or axial rotatory movement 16 to 27 days after sensitization (mean 21.3 days). Five of the 14 animals relapsed 2 to 6 wk after the first attack. Histological examination revealed inflammatory lesion, with significant demyelination in the central nervous system. Antibody levels to DM-20 were not related to the clinical signs. Five of 11 BALB/c nude mice reconstituted with T cells developed similar clinical and pathologic signs. This DM-20-induced EAE in mice may provide a valuable model because it is similar to multiple sclerosis and because it can be induced in inbred mice in which immune mechanisms can be easily studied.     

Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis

The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.     

Treatment of experimental allergic encephalomyelitis with myelin basic protein: Which route is best?

When myelin basic protein (BP) has been used for the treatment of multiple sclerosis (MS), it has been injected intramuscularly (IM) or subcutaneously (SC). Experimental allergic encephalomyelitis (EAE) is widely used as a model for MS, and the use of BP for MS is based on its efficacy in EAE. The present work shows that BP is more effective in EAE when administered by intravenous (IV) route than by IM or SC routes. These observations may be pertinent to therapeutic trials in MS.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Intercellular adhesion molecule-1 expression is required on multiple cell types for the development of experimental autoimmune encephalomyelitis

Many members of the Ig superfamily of adhesion molecules, such as ICAM-1 and VCAM-1, have been implicated in the pathogenesis of multiple sclerosis. Although it is well-established that VCAM-1/VLA-4 interactions can play important roles in mediating CNS inflammatory events in multiple sclerosis patients and during the development of experimental allergic encephalomyelitis (EAE), the contributions of ICAM-1 are poorly understood. This is due in large part to conflicting results from Ab inhibition studies and the observation of exacerbated EAE in ICAM-1 mutant mice that express a restricted set of ICAM-1 isoforms. To determine ICAM-1-mediated mechanisms in EAE, we analyzed ICAM-1 null mutant mice (ICAM-1(null)), which express no ICAM-1 isoforms. ICAM-1(null) mice had significantly attenuated EAE characterized by markedly reduced spinal cord T cell infiltration and IFN-gamma production by these cells. Adoptive transfer of Ag-restimulated T cells from wild-type to ICAM-1(null) mice or transfer of ICAM-1(null) Ag-restimulated T cells to control mice failed to induce EAE. ICAM-1(null) T cells also showed reduced proliferative capacity and substantially reduced levels of IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-12 compared with that of control T cells following myelin oligodendrocyte glycoprotein 35-55 restimulation in vitro. Our results indicate that ICAM-1 expression is critical on T cells and other cell types for the development of demyelinating disease and suggest that expression of VCAM-1 and other adhesion molecules cannot fully compensate for the loss of ICAM-1 during EAE development.     

Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab_26–37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.     

Dendritic cells induced in the presence of GM-CSF and IL-5

Experimental autoimmune encephalomyelitis (EAE) is commonly regarded as an animal model of the human disease multiple sclerosis (MS). Pertussis toxin (PTX) is routinely used for EAE induction in mice. Besides opening the blood-brain barrier, it acts as an adjuvant causing strong expansion of antigen-specific cells after coinjection with neuroantigens in IFA. Using an IL-17 ELISPOT assay we developed previously, we investigated the capability of PTX to induce proteolipid protein peptide 139-151(PLPp)-specific Th-17 cells in the immune periphery and in the thymus after coinjection with PLPp/IFA. PTX was found to induce peripheral PLPp-specific Th-17 cells in the draining lymph node and in the spleen, but not in the thymus. Our study indicates a new mechanism by which microbial agents can initiate or maintain autoimmune reactions and supports the growing role in particular for Th-17 cells in organ-specific autoimmune diseases like multiple sclerosis or EAE.     

Increased Spontaneous Apoptosis of Rat Primary Neurospheres In Vitro After Experimental Autoimmune Encephalomyelitis

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.     

Lipid microarrays identify key mediators of autoimmune brain inflammation

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.     

Analysis of protein induction in the CNS of SJL mice with experimental allergic encephalomyelitis by proteomic screening and immunohistochemistry.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by demyelination and inflammatory infiltrates in the CNS, and it is an animal model of multiple sclerosis. Piperonyl butoxide (PBO) suppresses disease in EAE mice, and it exhibits a dual effect on cytochrome P450s that manifests in a transient inhibitory phase followed by induction. In order to identify the expression of proteins associated with EAE, a proteomic screening was performed on hindbrain microsomes from control + vehicle, control + PBO, EAE + vehicle, and EAE + PBO female mice. Glucose regulated protein 94 (Grp94) and coagulation factor VIII were among the proteins identified in EAE + vehicle and EAE + PBO mice. Immunohistochemical staining of Grp94 was present in some neurons and oligodendrocytes in hindbrain sections from control animals, and in some cells within inflammatory infiltrates in EAE animals. Since Grp94 (also known as Gp96) can partake in antigen presentation and induction of proinflammatory cytokine expression, its presence in these cells suggests that it may play a role in the pathogenesis of EAE. Coagulation factor VIII is carried and protected by von Willebrand factor. Immunohistochemical staining of von Willebrand factor revealed its presence in some vessels within hindbrain sections from control animals. In EAE animals, the number of labeled vessels was significantly increased, and extracellular granular deposits were observed around labeled vessels indicating that the breakdown of the blood-brain barrier that occurs in EAE permitted its extravasation into the CNS. Additional proteins were identified in the different groups of mice by proteomic screening, but confirmation of their expression profile awaits investigations by independent measures.     

Proteome analysis of brain in murine experimental autoimmune encephalomyelitis

Multiple sclerosis is considered a prototype inflammatory autoimmune disorder of the CNS. Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein is one of the best‐characterized animal models of multiple sclerosis. Comprehensive understanding of gene expression in EAE can help identify genes that are important in drug response and pathogenesis. We applied a 2‐DE‐based proteomics approach to analyze the protein expression pattern of the brain in healthy and EAE samples. Of more than 1000 protein spots we analyzed, 70 showed reproducible and significant changes in EAE compared to controls. Of these, 42 protein spots could be identified using MALDI TOF‐TOF‐MS. They included mitochondrial and structural proteins as well as proteins involved in ionic and neurotransmitter release, blood barriers, apoptosis, and signal transduction. The possible role of these proteins in the responses of mice to animal models of multiple sclerosis is discussed.