PSGL‐1 and E/P‐selectins are essential for T‐cell rolling in inflamed CNS microvessels but dispensable for initiation of EAE

T‐cell migration across the blood‐brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P‐selectin glycoprotein ligand‐1 (PSGL‐1) and its endothelial ligands E‐ and P‐selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL‐1 or E/P‐selectins does not result in ameliorated EAE, we speculated that T‐cell entry into the spinal cord is independent of PSGL‐1 and E/P‐selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL‐1^?/? proteolipid protein‐specific T cells in inflamed spinal cord microvessels of WT or E/P‐selectin^?/? SJL/J mice during EAE. T‐cell rolling but not T‐cell capture was completely abrogated in the absence of either PSGL‐1 or E‐ and P‐selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T‐cell invasion into the CNS parenchyma. Thus, PSGL‐1 interaction with E/P‐selectin is essential for T‐cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T‐cell rolling is not required for successful T‐cell entry into the CNS and initiation of EAE.     

Mechanisms regulating regional localization of inflammation during CNS autoimmunity

Multiple sclerosis (MS) is a disease of the central nervous system (CNS) characterized by inflammatory, demyelinating lesions localized in the brain and spinal cord. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is induced by activating myelin-specific T cells and exhibits immune cell infiltrates in the CNS similar to those seen in MS. Both MS and EAE exhibit disease heterogeneity, reflecting variations in clinical course and localization of lesions within the CNS. Collectively, the differences seen in MS and EAE suggest that the brain and spinal cord function as unique microenvironments that respond differently to infiltrating immune cells. This review addresses the roles of the cytokines interferon-γ and interleukin-17 in determining the localization of inflammation to the brain or spinal cord in EAE.     

Low-level laser therapy ameliorates disease progression in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating inflammatory disease characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. The existing therapies for MS are only partially effective and are associated with undesirable side effects. Low-level laser therapy (LLLT) has been clinically used to treat inflammation, and to induce tissue healing and repair processes. However, there are no reports about the effects and mechanisms of LLLT in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here, we report the effects and underlying mechanisms of action of LLLT (AlGaInP, 660?nm and GaAs, 904?nm) irradiated on the spinal cord during EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG_35–55 peptide emulsified in complete Freund’s adjuvant. Our results showed that LLLT consistently reduced the clinical score of EAE and delayed the disease onset, and also prevented weight loss induced by immunization. Furthermore, these beneficial effects of LLLT seem to be associated with the down-regulation of NO levels in the CNS, although the treatment with LLLT failed to inhibit lipid peroxidation and restore antioxidant defense during EAE. Finally, histological analysis showed that LLLT blocked neuroinflammation through a reduction of inflammatory cells in the CNS, especially lymphocytes, as well as preventing demyelination in the spinal cord after EAE induction. Together, our results suggest the use of LLLT as a therapeutic application during autoimmune neuroinflammatory responses, such as MS.     

DAB389IL-2 suppresses autoimmune inflammation in the CNS and inhibits T cell-mediated lysis of glial target cells

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). DAB₃₈₉IL-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, DAB₃₈₉IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB₃₈₉IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB₃₈₉IL-2 that allowed substantial transmigration of inflammatory cells across the blood–brain barrier. DAB₃₈₉IL-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 DAB₃₈₉IL-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. DAB₃₈₉IL-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 DAB₃₈₉IL-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.     

CD49d/CD29‐integrin controls the accumulation of plasmacytoid dendritic cells into the CNS during neuroinflammation

Plasmacytoid dendritic cells (pDCs) are found in the CNS during neuroinflammation and have been reported to exert regulatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of entry of pDCs into the CNS as well as their phenotype and innate functional properties, once recruited into the CNS, have not been thoroughly examined. Herein, we show that pDCs rapidly accumulate into the brain and spinal cord during the acute phase of EAE, and maintain the expression of numerous phenotypic markers typical of peripheral pDCs. Functionally, CNS‐pDCs constitutively expressed IRF7 and were able to rapidly produce type I IFNs and IL‐12p40 upon ex vivo TLR‐9 stimulation. Using adoptive transfer experiments, we provide evidence that CNS‐pDC are recruited from the blood and accumulate into the CNS during the acute phase of EAE. Accumulation of pDCs into the CNS was strongly inhibited in the absence of CD29, but not CD18, suggesting a major role for ß1 but not ß2 integrins. Indeed, blocking the CD49d α4‐integrins during acute EAE drastically diminished CNS‐pDC numbers. Together, our results demonstrate that circulating pDCs are actively recruited into the CNS during acute EAE through a mechanism largely dependent on CD49d/CD29‐integrins.     

Antigen-specific down-regulation of myelin basic protein-reactive T cells during spontaneous recovery from experimental autoimmune encephalomyelitis: further evidence of apoptotic deletion of autoreactive T cells in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) was inducedin Lewis rats by the I.v. injection of 10⁷ cloned V_ß8.2⁺T cells specific for the 72–89 peptide of guinea pig myelinbasic protein(MBP). Some animals were injected simultaneouslywith 10⁷ cloned T cells specific for ovalbumin(OVA). Lymphocyteswere isolated from the spinal cord and from the peripheral lymphoidorgans of these rats and the frequencies of MBP-peptide-specificor OVA-specific proliferating cells were estimated by limitingdilution analysis at different times after cell transfer. Thefrequencies of cells specific for MBP_72–89 or OVA in thespinal cord were highest 5 days after cell transfer (MBP_72–89,1 in 1149; OVA, 1 in 1116). On day 7, when the rats were recovering,the frequency of cells specific for MBP_72–89 in the spinalcord fell dramatically to <1 in 10⁵, while that of OVA-specificcells decreased to a much lesser extent (1 in 7001). The frequenciesof MBP_72–89-specific cells in the peripheral lymphoidorgans during and after recovery were also much lower than thoseof OVA-specific cells. A similar pattern of down-regulationof the MBP-peptide-specific, but not the OVA-specific, T cellresponse was observed in the spleen and mesenteric lymph nodes(MLN) of rats 38 days after the active induction of EAE by immunizationwith equal amounts of MBP and OVA in adjuvants. In the passivelytransferred model, cells isolated from the spinal cord and MLNon day 7 did not regain responsiveness to MBP_72–89 afterincubation with high levels of IL-2, indicating that the unresponsivenesswas not due to T cell anergy. Thus this study demonstrates thatthere is a specific down-regulation of the MBP_72–89-specificT cell response during spontaneous recovery from EAE. This conclusionis consistent with our previous observation that V_ß8.2⁺T cells are selectively eliminated from the CNS by apoptosisduring recovery from EAE induced by the passive transfer ofV_ß8.2⁺ T cells reactive to this MBP peptide. In contrastto autoreactive T cells, the non-autoreactive T cells that accumulatein the CNS during EAE appear to recirculate to the peripherallymphoid organs.     

Calpain Activity in Spinal Cord Cells from Rats with Experimental Allergic Encephalomyelitis of Different Grades

The Ca²⁺-dependent calpain system of intracellular proteases is involved in regulating a multitude of physiological body functions. However, hyperactivation of calpains in cells is observed during the development of a number of responses, and this leads to impairment to the functioning of vitally important physiological systems. We report here our use of a model of experimental allergic encephalomyelitis (EAE) to demonstrate that cell homogenates from different parts of spinal cord show hyperactivation of calpains and that calpain proteolytic activity increases with the severity of EAE; this occurred not only in the lower, but also in the upper parts of the spinal cord. As CNS cells from animals with EAE showed increases in the levels of mRNA for m- but not for μ-calpain, this hyperactivation would appear to be mediated more by m-calpain. The relationship between the distribution of m-calpain hyperactivation in different parts of the spinal cord with the severity of EAE is consistent with data on the volume of neurological destruction of the CNS, suggesting that m-calpain is involved in the processes initiating neuron and cell death during the development of this disease.     

Inflammation and dephosphorylation of the tight junction protein occludin in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is a disease of the CNS in which inflammation, demyelination and neurodegeneration contribute to its initiation and progression. A frequently employed model of MS is experimental autoimmune encephalomyelitis (EAE). Here, to gain new insights into the disease process, an analysis of proteins in extracts of lumbar spinal cord from naïve and EAE rats was undertaken. The data mainly confirm that inflammation and blood–brain barrier (BBB) breakdown are the major hallmarks of disease in this model. Given their importance in the BBB, junctional proteins were further investigated. Occludin, a protein localizing to tight junctions in brain endothelial cells, showed strikingly increased migration in EAE when analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). This increased migration was mimicked by in vitro phosphatase treatment, implying its dephosphorylation in EAE. Occludin dephosphorylation coincided with the onset of inflammation, slightly preceding visible signs of disease, and was just prior to apparent changes in BBB permeability. These findings suggest occludin is a target for signaling processes in EAE, perhaps regulating the response of the BBB to the inflammatory environment as seen in MS.     

Role of cervical lymph nodes in autoimmune encephalomyelitis in the Lewis rat

Lymphocytes enter the central nervous system (CNS) in response to virus infections and in autoimmune diseases, such as multiple sclerosis (MS), but the origin of such lymphocytes is unclear. This study investigates the role of the cervical lymph nodes as a source of lymphocytes involved in experimental autoimmune disease of the brain. Acute active experimental autoimmune encephalomyelitis (EAE) is used as a model for the autoimmune aspects of MS and is characterized by lymphocyte and monocyte invasion and microglial activation, mainly in the spinal cord, 12–15 days post-inoculation (dpi) of antigen. Few lesions occur in the cerebral hemispheres in acute EAE, but a cryolesion to the surface of the brain 8 dpi results in a six-fold enhancement of cerebral EAE. The present study tests the hypothesis that cervical lymphadenectomy will reduce the enhancement of cerebral EAE induced by a cryolesion. Acute EAE was induced in 25 Lewis rats and a cryolesion to the brain, 8 dpi, in 16 rats was immediately followed by either cervical lymphadenectomy (n=8) or sham lymphadenectomy (n=8). The severity of EAE at 15 dpi, in the brain and spinal cord, was evaluated using immunocytochemistry for T lymphocytes (W3/13) and MHC class II expression (OX6). The results of the study showed that cervical lymphadenectomy reduced the level of cerebral EAE induced by a cryolesion by 40 per cent when compared with the sham-operated animals (P<0·01). This suggests that cervical lymph nodes play a pivotal role in the induction of EAE in the brain, possibly as a site for ‘priming’ T cells to target the brain. Investigation of the interrelationships between cervical lymph nodes and the brain in man may lead to new therapeutic strategies for multiple sclerosis. © 1997 John Wiley & Sons, Ltd.     

CCR1 antagonist J-113863 corrects the imbalance of pro- and anti-inflammatory cytokines in a SJL/J mouse model of relapsing-remitting multiple sclerosis

Multiple sclerosis (MS), an immune-mediated and neurodegenerative disorder of the central nervous system (CNS), is characterized by infiltrating myelin-reactive T lymphocytes and demyelinating lesions. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model used to study MS. To explore the impact of chemokine receptor CCR1 blockade in EAE and the underlying mechanisms, we used CCR1 antagonist J-113863 in PLP_139-151-induced EAE in SJL/J mice. Following EAE induction, mice were treated with J-113863 (10 mg/kg) daily from day 14 until day 25. We investigated the effect of J-113863 on expression levels of GM-CSF, IL-6, IL-10, IL-27 in CD4⁺ spleen cells, using flow cytometry. We also analyzed the effect of J-113863 on GM-CSF, IL-6, IL-10, IL-27 mRNA and protein expression levels using RT-PCR and Western blot analysis in brain tissues. J-113863 treatment decreased the populations of CD4⁺GM-CSF⁺ and CD4⁺IL-6⁺ cells and increased CD4⁺IL-27⁺ and CD4⁺IL-10⁺ cells in the spleen. J-113863 had a suppressive effect on the mRNA and protein expression levels of GM-CSF, and IL-6 in the brain tissue. On the other hand, J-113863 treatment increased the mRNA and protein expression of IL-10 and IL-27 in the brain tissue. Our results highlighted J-113863′s potential role in suppressing pro-inflammatory expression and up-regulating anti-inflammatory mediators, which could represent a beneficial alternative approach to MS treatment.     

Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice

Experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model (C57/BL6) to investigate the antioxidant status of animals at various clinical stages of the disease. For this purpose, blood, brain and spinal cord samples from EAE mice were collected and examined at different scores following post-immunization with myelin oligodendrocyte glycoprotein (MOG). The clinical sign of mobility of animals on different days was associated with gradual increase in lipid peroxidation products (malondialdehyde, i.e. MDA) in brain and spinal cord. Changes in lipid peroxidation during EAE progression was inversely related to superoxide dismutase (SOD) activity in erythrocyte preparation. However, suppression of catalase in erythrocytes, tissue glutathione (GSH) and plasma total antioxidant capacity (FRAP assay) were the early events in EAE, occurred during scores 1 and 2. Biochemical alterations were corroborated with histopathological observations showing demyelination and inflammatory foci in central nervous system (CNS) of animals suffering from partial hind limb paralysis (score 3). These data suggest that generation of MDA in CNS is a continuous process during EAE induction and suppression of antioxidant factors are early events of the disease, but crucial in increasing the vulnerability of CNS to demyelinating lesions.     

How myeloid cells shape experimental autoimmune encephalomyelitis: At the crossroads of outside-in immunity

Experimental autoimmune encephalomyelitis (EAE) is an animal model of central nervous system (CNS) autoimmunity. It is most commonly used to mimic aspects of multiple sclerosis (MS), a demyelinating disorder of the human brain and spinal cord. The innate immune response displays one of the core pathophysiological features linked to both the acute and chronic stages of MS. Hence, understanding and targeting the innate immune response is essential. Microglia and other CNS resident MUs, as well as infiltrating myeloid cells, diverge substantially in terms of both their biology and their roles in EAE. Recent advances in the field show that antigen presentation, as well as disease-propagating and regulatory interactions with lymphocytes, can be attributed to specific myeloid cell types and cell states in EAE lesions, following a distinct temporal pattern during disease initiation, propagation and recovery. Furthermore, single-cell techniques enable the assessment of characteristic proinflammatory as well as beneficial cell states, and identification of potential treatment targets. Here, we discuss the principles of EAE induction and protocols for varying experimental paradigms, the composition of the myeloid compartment of the CNS during health and disease, and systematically review effects on myeloid cells for therapeutic approaches in EAE.     

γδ T cells in EAE: Early trafficking events and cytokine requirements

We have previously shown that γδ T cells traffic to the CNS during EAE with concurrently increased expression of β₂‐integrins and production of IFN‐γ and TNF‐α. To extend these studies, we transferred bioluminescent γδ T cells to WT mice and followed their movement through the acute stages of disease. We found that γδ T cells rapidly migrated to the site of myelin oligodendrocyte glycoprotein peptide injection and underwent massive expansion. Within 6 days after EAE induction, bioluminescent γδ T cells were found in the spinal cord and brain, peaking in number between days 10 and 12 and then rapidly declining by day 15. Reconstitution of γδ T cell^?/? mice with γδ T cells derived from β₂‐integrin‐deficient mice (CD11a, ‐b or ‐c) demonstrated that γδ T‐cell trafficking to the CNS during EAE is independent of this family of adhesion molecules. We also examined the role of γδ T‐cell‐produced IFN‐γ and TNF‐α in EAE and found that production of both cytokines by γδ T cells was required for full development of EAE. These results indicate that γδ T cells are critical for the development of EAE and suggest a therapeutic target in demyelinating disease.     

MicroRNA signature of central nervous system‐infiltrating dendritic cells in an animal model of multiple sclerosis

Innate immune cells are integral to the pathogenesis of several diseases of the central nervous system (CNS), including multiple sclerosis (MS). Dendritic cells (DCs) are potent CD11c⁺ antigen‐presenting cells that are critical regulators of adaptive immune responses, particularly in autoimmune diseases such as MS. The regulation of DC function in both the periphery and CNS compartment has not been fully elucidated. One limitation to studying the role of CD11c⁺ DCs in the CNS is that microglia can upregulate CD11c during inflammation, making it challenging to distinguish bone marrow‐derived DCs (BMDCs) from microglia. Selective expression of microRNAs (miRNAs) has been shown to distinguish populations of innate cells and regulate their function within the CNS during neuro‐inflammation. Using the experimental autoimmune encephalomyelitis (EAE) murine model of MS, we characterized the expression of miRNAs in CD11c⁺ cells using a non‐biased murine array. Several miRNAs, including miR‐31, were enriched in CD11c⁺ cells within the CNS during EAE, but not LysM⁺ microglia. Moreover, to distinguish CD11c⁺ DCs from microglia that upregulate CD11c, we generated bone marrow chimeras and found that miR‐31 expression was specific to BMDCs. Interestingly, miR‐31‐binding sites were enriched in mRNAs downregulated in BMDCs that migrated into the CNS, and a subset was confirmed to be regulated by miR‐31. Finally, miR‐31 was elevated in DCs migrating through an in vitro blood–brain barrier. Our findings suggest miRNAs, including miR‐31, may regulate entry of DCs into the CNS during EAE, and could potentially represent therapeutic targets for CNS autoimmune diseases such as MS.     

Allergic encephalomyelitis: immunologically specific inhibition of cellular passive transfer by encephalitogenic basic proteins

Passive transfer of experimental allergic encephalomyelitis (EAE) was performed with lymph node cells from donor rats immunized with spinal cord or purified encephalitogenic basic protein and any of several adjuvant combinations. Transfer was inhibited by injection of recipients with the brain basic protein. Basic proteins from rat and guinea-pig CNS inhibited transfer from donors immunized with rat or guinea-pig spinal cord or basic protein. Monkey basic protein inhibited transfer from donors immunized with monkey or human cord. There was only partial cross-inhibition between rodent and primate groups. The inhibition was organ specific in that basic proteins from lung, spleen and adrenal did not inhibit transfer of EAE. Furthermore, transfer of adrenalitis from donors immunized with adrenal tissue was inhibited by adrenal extracts but not by CNS basic protein.

Pertussis and typhoid vaccines injected into recipients inhibited transfer of either EAE or adrenalitis. This non-specific inhibition was also demonstrated after simultaneous transfer of both EAE and adrenalitis, whereas CNS basic protein eliminated only EAE in such recipients.

Inhibition was complete in experiments terminated one day after transfer. In longer experiments, inhibition lasted only 3 or 4 days. Basic protein was rapidly cleared from the blood. Neither spleen nor adrenals were necessary for its action. Brain basic protein prevented the interaction between encephalitogenic cells and the target organ by an immunologically specific mechanism which may be a type of desensitization.

The inhibition could not be reproduced in vitro. Bioassay and radioactive tracers revealed that basic protein bound non-specifically to living or dead lymph node cells in vitro. The effects of incubation could be accounted for by this non-specific carry-over of basic protein into the recipients.

     

1,25‐dihydroxyvitamin D3‐induced dendritic cells suppress experimental autoimmune encephalomyelitis by increasing proportions of the regulatory lymphocytes and reducing T helper type 1 and type 17 cells

Dendritic cells (DCs), a bridge for innate and adaptive immune responses, play a key role in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Administration of tolerogenic DCs has been used as an immunotherapy in autoimmune diseases. Deficiency of vitamin D is an environmental risk factor of MS. In this study, we induced tolerogenic DCs by 1,25‐dihydroxyvitamin D₃ and transferred the tolerogenic DCs (VD₃‐DCs) into EAE mice by adoptive transfer. We found that VD₃‐DCs inhibited the infiltrations of T helper type 1 (Th1) and Th17 cells into spinal cord and increased the proportions of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺), CD4⁺ IL‐10⁺ T cells and regulatory B cells (CD19⁺ CD5⁺ CD1d⁺) in peripheral immune organs, which resulted in attenuated EAE. However, the proportions of T helper type 1 (Th1) and Th17 cells in spleen and lymph nodes and the levels of pro‐inflammatory cytokines and IgG in serum also increased after transfer of VD₃‐DCs. We conclude that transfer of VD₃‐DCs suppressed EAE by increasing proportions of regulatory T cells, CD4⁺ IL‐10⁺ T cells and regulatory B cells in spleen and reducing infiltration of Th1 and Th17 cells into spinal cord, which suggests a possible immunotherapy method using VD₃‐DCs in MS.     

Kinetics of proinflammatory monocytes in a model of multiple sclerosis and its perturbation by laquinimod

Proinflammatory circulating monocytes have important roles in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Yet there is limited information on their accumulation in blood during disease, the mechanisms that regulate their infiltration into the central nervous system (CNS), and whether medications affect their biology. We found a significant and prolonged elevation of CD11b(+)CCR2(+)Ly6C(high) proinflammatory monocytes in the blood of mice by the second day of immunization for EAE. At onset of clinical signs, levels of proinflammatory monocytes plummeted to those in naive mice. At day 16, when the majority of mice were at peak disease severity, clinical scores were inversely correlated to the proportion of proinflammatory monocytes in blood, and directly correlated with that in the spinal cord. Treatment with the MS medication laquinimod prevented EAE, correspondent with retention of proinflammatory monocytes in blood. The reduced entry of proinflammatory monocytes into the CNS by laquinimod was attributed to reduction of their levels of CD62L and matrix metalloproteinase-9. Moreover, the spinal cord of laquinimod-treated mice did not have elevated levels of CCR2 and CCL2, which provide chemotactic cues for monocytes. These results shed light on the important role of the trafficking of proinflammatory monocytes into the CNS to promote disease activity, and they identify a mechanism of action of laquinimod in MS.     

Correlation of nitric oxide levels in the cerebellum and spinal cord of experimental autoimmune encephalomyelitis rats with clinical symptoms

Experimental autoimmune encephalomyelitis (EAE) is a well-established cell-mediated autoimmune inflammatory disease of the CNS, which has been used as a model of the human demyelinating disease. EAE is characterized by infiltration of the CNS by lymphocytes and mononuclear cells, microglial and astrocytic hypertrophy, and demyelination which cumulatively contribute to clinical expression of the disease. EAE was induced in female Sprague-Dawley rats, 3 months old (300 g ± 20 g), by immunization with myelin basic protein (MBP) in combination with Complete Freund's adjuvant (CFA). The animals were divided into 7 groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-L-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the level of nitric oxide (NO(·)) production was determined by measuring nitrite and nitrate concentrations in 10% homogenate of cerebellum and spinal cord. Obtained results showed that the level of NO(·) was significantly increased in all examined tissues of the EAE rats compared to the control and CFA groups. Also, AG and NAC treatment decreased the level of NO(·) in all tissues compared to the EAE group. The level of NO(·) is increased significantly in the spinal cord compared to the cerebellum. The clinical course of the EAE was significantly decreased in the EAE groups treated with AG and NAC during the development of the disease compared to EAE group and its correlates with the NO(·) level in cerebellum and spinal cord. The findings of our work suggest that NO(·) and its derivatives play an important role in multiple sclerosis (MS). It may be the best target for new therapies in human demyelinating disease and recommend the new therapeutic approaches based on a decreased level of NO(·) during the course of MS.     

Preferential distribution of Vβ 8.2-positive T cells in the central nervous system of rats with myelin basic protein-induced autoimmune encephalomyelitis

To determine the role of encephalitogenic T cells in the formation of lesions in the central nervous system (CNS), experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by immunization with either myelin basic protein (MBP) or the synthetic peptide which corresponds to the 87–100 sequence of guinea pig MBP, and T cells expressing T cell receptor (TcR) Vβ8.2, Vβ8.5, Vβ10 and Vβ16 in the lymphoid organs and CNS were localized and quantified by flow cytometry (FCM) and immunohistochemistry. In normal rats, the percentage of T cells expressing these Vβ phenotypes to the total number of TcR αβ⁺ T cells, as determined by FCM, ranged from 5% to 10% in the lymph node. Vβi6⁺ T cells were the most predominant population among the four Vβ subsets tested. Essentially the same findings were obtained from the analysis of the lymphoid organs of rats with EAE which had been induced by immunization with the same two antigens. In sharp contrast, 15–20% of the T cells isolated from lesions of MBP-induced EAE expressed Vβ8.2⁺. Thus, the percentage of Vβ8.2⁺ T cells in the EAE lesions was threefold higher than that in the lymph node, while the proportions of Vβ8.5⁺, Vβ10⁺ and Vβ16⁺ T cells were about the same in both organs. The predominance of Vβ58.2⁺ T cells in EAE lesions was confirmed by counts of immunohistochemically stained T cells in the spinal cord. Moreover, it was revealed that (i) the predominance of Vβ8.2⁺ T cells was greatest during the development of EAE and became less obvious at the recovery stage, and (ii) at the peak stage of EAE, approximately 85% of Vβ8.2⁺ T cells were distributed in the parenchyma while 15% were in the perivascular space of the CNS vessels. These findings indicate that encephalitogenic T cells which express Vβ8.2 infiltrate the CNS at a very early stage of EAE and become the predominant population in infiltrating T cells, and further suggest that encephalitogenic T cells, not only recruit inflammatory cells in the CNS, but also cause neural tissue damage, such as demyelination.