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In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated CD4(+) T cells with upregulated IL-2R mediate inflammation and demyelination in the central nervous system (CNS). DAB(389)IL-2, a chimeric fusion protein of IL-2 and diphtheria toxin, inhibits human and rodent IL-2 activated T cells that express the high affinity interleukin-2 receptor. In the present study, DAB(389)IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB(389)IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB(389)IL-2 that allowed substantial transmigration of inflammatory cells across the blood-brain barrier. DAB(389)IL-2 inhibited infiltration of CD4(+), CD8(+), CD25(+) and TCR αβ(+) associated mononuclear cells and inflammatory macrophages in the spinal cord on day 13 post-immunization, at the peak of disease. Gene expression study showed that DAB(389)IL-2 treatment suppressed TNF-α and IFN-γ as well as IL-10 cytokine gene expression in the spinal cord of rats with EAE on day 13. DAB(389)IL-2 in vitro treatment suppressed cytotoxicity of MBP-activated T cells from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated T cells in vitro. This study suggests that DAB(389)IL-2 directly targets CD4(+) and CD25(+) (IL-2R) T cells and effector T cell function and also indirectly suppresses the activation of macrophage CD169(+) (ED3(+)) and microglia CD11b/c (OX42(+)) populations in the CNS., (© 2013.)

Background: Neuronal loss in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), correlates with permanent neurological dysfunction. Current MS therapies have limited the ability to prevent neuronal damage., Methods: We examined whether oral therapy with SRT501, a pharmaceutical grade formulation of resveratrol, reduces neuronal loss during relapsing-remitting EAE. Resveratrol activates SIRT1, an NAD+-dependent deacetylase that promotes mitochondrial function., Results: Oral SRT501 prevented neuronal loss during optic neuritis, an inflammatory optic nerve lesion in MS and EAE. SRT501 also suppressed neurological dysfunction during EAE remission, and spinal cords from SRT501-treated mice had significantly higher axonal density than vehicle-treated mice. Similar neuroprotection was mediated by SRT1720, another SIRT1-activating compound; and sirtinol, an SIRT1 inhibitor, attenuated SRT501 neuroprotective effects. SIRT1 activators did not prevent inflammation., Conclusions: These studies demonstrate that SRT501 attenuates neuronal damage and neurological dysfunction in EAE by a mechanism involving SIRT1 activation. SIRT1 activators are a potential oral therapy in MS.

Purpose: Acute vision loss from optic neuritis typically resolves; however, recovery is often not complete. Permanent vision loss from retinal ganglion cell (RGC) death occurs in 40% to 60% of patients. Current therapy (high-dose corticosteroids) speeds recovery but does not change final visual outcomes. Here the authors examined whether corticosteroids administered early in the disease course can prevent RGC loss in experimental optic neuritis., Methods: RGCs were retrogradely labeled with fluorogold in SJL/J mice. Experimental autoimmune encephalomyelitis (EAE) was induced by immunization with proteolipid protein peptide. Optic neuritis began 9 days after immunization. Mice were treated daily with dexamethasone, methylprednisolone, or PBS from days 0 to 14 or days 10 to 14 and then were killed on day 14, 18, or 22., Results: Corticosteroid treatment initiated before optic neuritis onset (days 0-14) suppressed EAE and reduced optic neuritis incidence through day 14. In the few eyes that developed optic neuritis, inflammation was mild, and RGC loss was attenuated. After treatment was stopped on day 14, mice rapidly developed EAE and optic neuritis by day 18, but RGC loss was still reduced. By day 22, RGC loss increased to levels similar to those of untreated optic neuritis eyes. Corticosteroid treatment after optic neuritis onset (days 10-14) slowed EAE progression and showed a trend toward suppression of optic neuritis and RGC loss on day 14 that was lost by day 18., Conclusions: Corticosteroids can suppress optic neuritis and prevent RGC loss if treatment is initiated before optic nerve inflammation onset. Treatment is less effective after inflammation begins. Results suggest that chronic immunomodulation may prevent recurrent optic neuritis and RGC damage.

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is an extract enriched with BBI, but predominantly contains other ingredients including several protease inhibitors. We previously found that BBIC administration to Lewis rats with experimental autoimmune encephalomyelitis (EAE) significantly suppresses disease. In the present study we determined whether BBI mediates the suppressive effects of BBIC in EAE, evaluated its potential neuroprotective effects, and investigated mechanisms of BBI action. We tested effects of purified BBI on clinical and histopathological parameters of EAE in two models (relapsing/remitting EAE in SJL/J mice and chronic EAE in C57BL/6 mice). Effects of BBI were compared to BBIC in relapsing/remitting EAE, and effects of BBI on neuronal survival were examined during acute optic neuritis. Treatment with BBI in both EAE models significantly improved EAE disease parameters (onset, severity, weight loss, inflammation and demyelination). BBI significantly reduced the incidence of optic neuritis and prevented loss of retinal ganglion cells. In most experiments proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. Using Boyden's chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. BBI also induced higher production of EAE-suppressive cytokine IL-10 by immune cells. These results demonstrate that BBI is the active component of BBIC that ameliorates clinical EAE. BBI reduces inflammation and attenuates neuronal loss, making it an excellent candidate for oral therapy in MS. BBI likely ameliorates EAE by inhibiting multiple pathways involved in disease pathogenesis.

Activated T cells express the high affinity interleukin 2 receptor (IL-2R also CD25) that binds interleukin 2 (IL-2) and transduces signals important for the proliferation and survival of these cells. We investigated the effect of the genetically engineered immunotoxin DAB(389)IL-2 on experimental autoimmune encephalomyelitis (EAE), an autoimmune disease of the central nervous system (CNS) mediated by activated myelin-reactive T cells. EAE is the most commonly used animal model of the human disease multiple sclerosis (MS). DAB(389)IL-2 is a recombinant fusion product made of a portion of diphtheria toxin, which contains binding and translocation components of the toxin linked to IL-2. The diphtheria toxin targets and kills cells expressing the high affinity IL-2 receptor and has been successfully used in several autoimmune and neoplastic conditions. We observed a significant suppression of guinea-pig spinal cord homogenate (gpSCH)-MBP induced active EAE in Lewis rats at 2 x 1,600 kU of DAB(389)IL-2 given on days 7 and 9 post-immunization and complete suppression with the same dose on days 7, 8 and 9 or 7, 8, 9 and 10 after immunization during the active disease period. There were reduced mononuclear cell infiltrates of CD4(+), CD8(+), CD25(+) and alphabetaTCR(+) T cells in the spinal cord of treated rats. However, treatment at day 11 or 12 post-immunization led to severe, fatal disease. The toxin added to cultures in vitro or injected in vivo suppressed antigen- and mitogen-induced T cell proliferation. DAB(389)IL-2 treatment in vivo or exposure of encephalitogenic T cells in vitro prior to transfer did have a significant inhibitory effect on adoptive transfer EAE. Our data demonstrate that DAB(389)IL-2 immunotoxin can suppress active and passive EAE if applied at specific, early time points, but can have negative consequences at later time points.

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is mediated by autoantigen-specific T-helper1 (Th1) cells. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects in EAE. Programmed death-1 (PD-1) and PD-1 ligand (PD-L), new members of the B7 superfamily of costimulatory molecules, play a critical role in regulating EAE. Whether the interaction of IL-12 and the PD-1/PD-L pathway regulates EAE is unclear. We have previously shown that IL-12 suppresses EAE induced by MOG35-55 in C57BL/6 mice, but not in IFN-gamma-deficient mice, suggesting that IFN-gamma is required for the inhibitory effects of IL-12 on EAE. In the current study, PD-L1 expression is up-regulated following IL-12 treatment in wild-type mice, but not in IFN-(-deficient EAE mice. Similarly, IL-12 induces IFN-gamma and PD-L1 expression in cultured MOG-specific T cells from wild-type mice but not from IFN-gamma-deficient mice. Furthermore, PD-L1 expression increased specifically in CD11b+ antigen presenting cells (APCs) after IL-12 administration. These data suggest that one mechanism of IL-12 suppression of EAE is mediated by PD-1/PD-L signaling downstream of IFN-gamma induction in CD11b+ APCs. The regulation of PD-1/PD-L1 may have potential therapeutic effects for EAE and MS.

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

To characterize T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis (RR-EAE), we compared myelin oligodendrocyte glycoprotein (MOG)-induced RR-EAE in C57BL/6 (B6) x SJL (F1) mice and chronic-progressive EAE (CP-EAE) in B6 mice at week 8 p.i. when clinical scores were comparable. Although these two strains exhibited similar inflammation/demyelination pattern and MOG-induced T cell responses, RR-EAE mice produced significantly higher levels of anti-MOG IgG1/IgG2a antibodies. Further, lymphocytes of RR-EAE mice proliferated vigorously to the secondary epitope myelin basic protein (MBP) 1-11. These results support a potential involvement of anti-MOG antibodies and epitope spreading in T cell responses in the development of MOG-induced RR-EAE model.

IL-12 was thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. However, we have recently found that IL-12 responsiveness, via IL-12Rbeta2, is not required in the induction of EAE. To determine the role of IL-12Rbeta1, a key subunit for the responsiveness to both IL-12 and IL-23, in the development of autoimmune diseases, we studied EAE in mice deficient in this subunit of IL-12R. IL-12Rbeta1(-/-) mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th2 response. To study the mechanism underlying this Th2 bias, we cocultured purified CD4(+) T cells and APCs of MOG-immunized mice. We demonstrate that IL-12Rbeta1(-/-) APCs drive CD4(+) T cells of both wild-type and IL-12Rbeta1(-/-) mice to an Ag-induced Th2 phenotype, whereas wild-type APCs drive these CD4(+) T cells toward a Th1 type. IL-12Rbeta1(-/-) CD4(+) T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-gamma and TNF-alpha. Furthermore, decreased levels of IL-12p40, p35, and IL-23p19 mRNA expression were found in IL-12Rbeta1(-/-) APCs, indicating an autocrine pathway of IL-12/IL-23 via IL-12Rbeta1. IL-18 production and IL-18Ralpha expression are also significantly decreased in IL-12Rbeta1(-/-) mice immunized with MOG. We conclude that in the absence of IL-12Rbeta1, APCs play a prominent regulatory role in the induction of autoantigen-specific Th2 cells.

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

Experimental allergic encephalomyelitis (EAE) can be downregulated by intravenous (iv) administration of myelin basic protein (MBP). In this report we show that downregulation of EAE by two 500-microgram doses of MBP administered iv before immunization was associated with reduced encephalitogenicity of both spleen and lymph node cells (day 12 postimmunization) in adoptive transfer studies. However, efficient downregulation of EAE by two 500-microgram iv doses of MBP on days 10 and 11 after active immunization (at the time of disease onset) was associated with no significant change in the encephalitogenicity of lymph node cells, but a complete abrogation of the ability of spleen cells (both at day 12 postimmunization) to transfer EAE compared to controls. Furthermore, coculture of spleen cells from rats tolerized by iv MBP on days 10 and 11 after active immunization with MBP with MBP-reactive T cells resulted in a decreased ability of the spleen T cells to transfer EAE compared to effector cells in monoculture. In contrast, coculture of MBP-reactive T cells with spleen cells from rats tolerized by iv MBP on days 14 and 7 before active immunization resulted in increased disease in recipient rats. These results suggest that reversal of clinical EAE by iv injection of MBP at the time of disease onset is due at least in part to a T cell control mechanism located in the spleen and suggest the presence of splenocyte regulatory cells that can suppress the ability of encephalitogenic T cells to induce EAE., (Copyright 2000 Academic Press.)

Blockade of the CD40 ligand (CD40L)-CD40 interaction suppresses experimental autoimmune encephalomyelitis (EAE). Since this interaction induces IL-12, an essential cytokine for EAE induction, we hypothesized that CD40L blockade may suppress EAE through IL-12 inhibition. Here we show that exogenous IL-12 abolishes the ability of anti-CD40L monoclonal antibodies to prevent EAE. Anti-IL-12 antibodies prevent this reversal and protect from EAE. These results show that IL-12 is sufficient to overcome CD40L blockade and suggest that, of the multiple consequences of the CD40L-CD40 interaction, IL-12 induction is an essential one for induction of EAE.

Immunological tolerance and suppression of clinical and histological experimental allergic encaphalomyelitis (EAE) can be induced by the intravenous (i.v.) administration of myelin basic protein (MBP). In this report we have characterized the effect of the time of i.v. administration of MBP on the course of EAE in Lewis rats. Rats were treated with the i.v. administration of one or two 500 micrograms doses of MBP either before or after active immunization. Results indicated that i.v. administration of MBP in rats before active immunization with MBP/CFA (naïve rats) was most effective when given 14 days before active immunization, but treatment of rats actively immunized with MBP (immunized rats) was most effective at the onset of disease. Treatment at other times was less effective. The i.v. administration of the peptide MBP 68-88 (p68-88) containing the dominant encephalitogenic epitope could also suppress MBP-induced EAE in a dose dependent manner. Intravenous administration of two injections of p68-88 to naïve rats on days 10 and 3 before, or on days 0 and 7 after, active immunization with MBP suppressed the development of EAE in a dose dependent manner. Treatment of rats with i.v. MBP after, but not before, the transfer of MBP-reactive EAE effector cells suppressed the development of EAE in the recipient rats. Transfer of lymphoid cells from tolerized naïve rats failed to protect recipient rats against development of active or passive EAE. These results indicate the importance of timing and dose of the antigen on the induction of tolerance and suggests different mechanisms of tolerance induction by intravenous MBP in immunized and naïve rats. They also emphasize the importance of timing in designing efficient treatment strategies when i.v. tolerance is contemplated in EAE and possibly multiple sclerosis.

Autoimmune encephalomyelitis can be initiated spontaneously and developed progressively in TCR transgenic mice specific for myelin basic protein when exposed to non-sterile environment, thus more closely mimicking human multiple sclerosis. By intravenous administration of myelin basic protein, we succeeded in reversing the clinical and pathological signs of progressive spontaneous disease in these mice. Flow cytometry showed that the majority of transgenic T cells in lymph nodes and spleen as well as spinal cords of treated mice were deleted. Dramatically increased numbers of apoptotic cells were found in peripheral immune organs of treated animals. Proliferative responses of single transgenic T cell to autoantigen were significantly decreased in treated mice, indicating that the remaining T cells were anergic. Moreover, production of both Th1 and Th2 cytokines was suppressed. This study is the first demonstration of reversal of progressive, spontaneous autoimmune disease of the central nervous system, and provides direct evidence that apoptosis-induced clonal deletion, along with anergy of remaining cells, but not Th2 switch, play a major part in the reversal of this disease by intravenous administration of autoantigen.

Immunization of (PL/J x SJL/J)F1 mice with myelin basic protein (MBP) induces relapsing experimental autoimmune encephalomyelitis (EAE). Relapses occur 7 to 10 days after recovery from the initial paralysis. Staphylococcal enterotoxins (SE) A or B, administered after recovery from the initial paralysis, induce immediate relapses. IL-12 is involved in the induction of EAE. Here, we show that SEA and SEB induce IL-12 in splenocytes from (PL/J x SJL/J)F1 mice in vitro and increase the level of IL-12 in the sera of mice treated with these superantigens. IL-12 administration mimics SE in inducing spontaneous relapses and in enhancing the severity and frequency of spontaneous relapses. IL-12 neutralization blocks SE-induced and subsequent relapses of EAE, and, when instituted after recovery from the initial attack, prevents spontaneous relapse. This is the first report of prevention of relapses of EAE with anti-IL-12 Ab, an approach which may prove useful in the prevention of exacerbations in multiple sclerosis.

Melatonin has immune-enhancing effects and can exacerbate autoimmunity. Pinealectomy or light exposure, which suppress melatonin, inhibit T cell autoimmunity. To investigate the involvement of melatonin in experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune demyelinating disease, we tested the effect of luzindole, a melatonin receptor antagonist, on EAE. Luzindole-treated mice did not develop EAE after immunization with spinal cord homogenate, whereas control mice developed EAE. This study suggests that pharmacological inhibition of the immunoenhancing effects of melatonin may prevent autoimmune demyelination.

Angiotensin converting enzyme (ACE)1 mediates inflammation, participates in T cell stimulation by certain antigenic peptides, and influences the permeability of the blood brain barrier (BBB). ACE is elevated in multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS), characterized by increased BBB permeability. ACE inhibitor captopril suppresses certain immune functions and inhibits inflammatory or autoimmune diseases. We studied the effect of captopril on Lewis rat EAE, an animal model of MS. Fourteen rats with EAE were treated with captopril 30 mg/kg daily from immunization to day 21 post-immunization, and compared with 14 untreated rats. Severity scores and lymphocyte reactivity to myelin basic protein and mitogen were measured. There was a statistically significant (p < 0.05) difference between the mean and cumulative clinical scores of captopril-treated and untreated animals. Lymphocytes from captopril treated EAE rats at the peak of disease severity had diminished responses to MBP and concanavalin A. The data suggest a significant beneficial effect of captopril in Lewis rat EAE. Further studies including other inhibitors of ACE or of other peptidases with immune, inflammatory or BBB role, may identify potentially valuable immunopharmacologic agents.