FasL诱导EAE小鼠淋巴细胞凋亡的实验研究

为了解FasL在诱导EAE淋巴细胞凋亡中发挥的作用 ,我们用髓鞘碱性蛋白 (MBP )致敏小鼠 ,建立动物模型 实验性自身免疫性脑脊髓膜炎 (EAE )。分析了EAE动物淋巴细胞膜上Fas及FasL分子的表达水平 ,检测了分泌的细胞因子 ;用FasL分子诱导了EAE淋巴细胞凋亡。结果表明 ,应用MBP致敏KM小鼠 ,成功地建立了EAE模型 ,KM小鼠的发病率为6 8 3% ,发病程度为 1～ 2分 ;小鼠特异性淋转比对照组高 ,并与发病评分成正比 ;细胞因子分泌水平提示以Th2为主 ;EAE淋巴细胞Fas和FasL的表达及凋亡显著高于对照组 ;FasL诱导淋巴细胞凋亡剂量依赖曲线表明 ,在一个较小的浓度范围内呈现正相关 ,若加入单抗可部分阻断FasL诱导的凋亡。结果提示Fas FasL在EAE动物淋巴细胞凋亡中起了重要作用。     

Gene Therapy in Experimental Autoimmune Encephalomyelitis

Gene therapy traditionally has been associated with gene replacement, where exogenous recombinant DNA is introduced ex vivo into somatic cells that are then introduced back into the patient as a way to correct an inherited genetic defect. However, several novel gene therapy strategies for treating autoimmune diseases recently have emerged. Strategies involving the use of several types of DNA vaccines, the application of various viral vectors, and the use of diverse cellular vectors have shown promise in inhibiting autoimmune-mediated inflammation and repairing tissue damaged as a result of autoimmune attack. In the current review, we examine and discuss the development and proposed use of emerging gene therapy strategies for the treatment of autoimmune disease with specific emphasis on experimental autoimmune encephalomyelitis (EAE), an animal model widely used in multiple sclerosis (MS) research.     

Netrin-1对脐静脉内皮细胞增殖及迁移的影响

目的：研究神经轴突导向因子Netrin-1对血管内皮细胞增殖和迁移的影响及其机制。方法：原代培养人脐静脉内皮细胞（HUVECs），分别用CCK-8和迁移实验检测Netrin-1对HUVECs增殖、迁移的作用。检测HUVECs上各Netrin-1受体的表达，用siRNA沉默HUVECs上该受体，观察增殖、迁移能力的改变。结果：Netrin-1对HUVECs的增殖和迁移有促进和抑制的双重导向作用。沉默受体UNC5B后，Netrin-1对于HUVECs的增殖和迁移的促进作用增强，而抑制作用消失。结论：Netrin-1对HUVECs的增殖和迁移具有浓度依赖性双重导向作用，其中抑制作用由UNC5B受体介导。     

Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.     

ICOS Deficiency Results in Exacerbated IL-17 Mediated Experimental Autoimmune Encephalomyelitis

Introduction  Inducible costimulatory molecule (ICOS) is important for the effector function of T cells, especially for Th2 and T cell dependent B cell responses. However, it has been shown that ICOS is required for the differentiation of Th17 cells. Since IL-17 has been identified as a major cytokine involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the enhanced severity of EAE in ICOS-deficient mice (ICOS^−/−) mice is unexpected. Methods  To better understand the role of ICOS and of IL-17 in EAE, we induced EAE in ICOS^−/− by immunization with myelin oligodendrocyte glycoprotein peptide (MOG_35–55) in complete Freund’s adjuvant. Results  As previously reported, we found that ICOS^−/− mice developed more severe EAE. Upon restimulation with MOG_35–55, splenocytes from ICOS^−/− mice with EAE produced higher amounts of IL-17 and ICOS^−/− mice had a higher expression of IL-17, IL-6, and TGF-β mRNA in the spinal cords at the onset of the disease. Finally, the blockade of IL-17 strongly inhibited disease even in ICOS^−/− mice, showing that IL-17 is playing a major role in the pathogenesis of EAE both in WT and ICOS^−/− mice. Conclusion  In conclusion, MOG immunization induces MOG-specific Th17 cells also in ICOS^−/− mice, and a higher expression of IL-17 and of Th17-driving cytokines IL-6 and TGF-β in the central nervous system at the onset of EAE that correlates with their more severe disease.     

Changes in the Neuronal Population of the Spinal Cord of Mice with Experimental Autoimmune Encephalomyelitis as a Model of Multiple Sclerosis

Bulletin of Experimental Biology and Medicine - We performed a quantitative study of the neuronal population in the spinal cord of mice with acute and chronic model of experimental autoimmune...     

The Relationship Between Aquaporin‐4 Expression and Blood‐Brain and Spinal Cord Barrier Permeability Following Experimental Autoimmune Encephalomyelitis in the Rat

Aquaporin 4(AQP4) is a water channel protein strongly expressed in the central nervous system in perimicrovessel astrocyte foot processes, the glia limitans, and ependyma. Expression of AQP4 is highest at the blood‐brain barrier and blood‐spinal cord barrier, supporting its critical function in material transport across these structures. Recently, presence of the anti‐aquaporin‐4 antibody in sera has been used as an important diagnostic tool for neuromyelitis optica, suggesting a potential role in central nervous system inflammation. The aim of the present study was to examine AQP4 protein expression in the cerebellum and spinal cord from rats with experimental autoimmune encephalomyelitis. By western blot analysis, AQP4 expression increased during experimental autoimmune encephalomyelitis development, and peaked at onset (lumbar enlargement) or climax (cerebellum) of neurological signs of experimental autoimmune encephalomyelitis. There was also a faster and more pronounced increase in permeability in the cerebellar blood‐brain barrier and the lumbar enlargement blood‐spinal cord barrier consistent with AQP4 expression, which was manifested by increased Evans Blue leakage and reduced tight junction protein expression. In conclusion, aquaporin upregulation may be involved in the development of inflammation in the acute phase of experimental autoimmune encephalomyelitis, and may correlate with damage to central nervous system barrier function. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The Effects of Pregnancy on Myelin Basic Protein-induced Experimental Autoimmune Encephalomyelitis in Lewis Rats: Suppression of Clinical Disease, Modulation of Cytokine Expression in the Spinal Cord Inflammatory Infiltrate and Suppression of Lymphocyte Proliferation by Pregnancy Sera

PROBLEM: The present study was performed to explore the effects of pregnancy on experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by inoculation with myelin basic protein (MBP) (MBP-EAE). METHOD OF STUDY: MBP-EAE was induced in pregnant and non-pregnant rats and severity of disease evaluated. Serum from pregnant and non-pregnant rats was used in standard lymphocyte proliferation assays. Real-time polymerase chain reaction (PCR) was used to investigate the expression of cytokine mRNA in the inflammatory cells obtained from the spinal cord of rats on day 15 after inoculation. RESULTS: Pregnant rats developed less severe disease than non-pregnant rats. Serum from pregnant rats suppressed the proliferation of T lymphocytes in response to MBP. There was significantly increased expression of IL-4, IL-10 and TNF-alpha mRNA in the spinal cord infiltrate of pregnant rats. CONCLUSION: Circulating humoral factors and alteration in cytokine production by inflammatory cells may contribute to the suppression of EAE in pregnant rats.     

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.     

Interaction between Gonadal Steroids and Neuroimmune System in Acute Experimental Autoimmune Encephalomyelitis (EAE) in Wistar Rats

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T lymphocytes directed against myelin antigens. Since neuroendocrine-immune dysfunction appears to contribute to the pathogenesis of autoimmune diseases, the present work was designed to study the effect of changes in the endocrine system on the development of acute EAE and the immune response against myelin basic protein (MBP). Intact and sham males and intact female Wistar rats showed the most severe clinical symptoms (acute period) 12–14 days post-inoculation (dpi). Then, they began gradually to recover, regaining the total ability to walk by 15–17 dpi. Male Wistar rats with altered levels of gonadal hormones by surgical castration showed an onset of the symptoms retarded 2–3 days with respect to the other EAE groups, showing neuropathological symptoms up to 27–28 dpi, and remaining with lower body weight even at 40 dpi. The castrated animals exhibited a specific delay in MBP-stimulated DTH reactivity that correlates with the delay in the onset of the clinical symptoms. Also significant lymphocyte proliferation to MBP was still present at 35 dpi that was absent in the sham group. The distribution of the IgG subclasses indicated that at 35 dpi castrated animals have a higher IgG2b/IgG1 ratio (35.1) in comparison to that presented by sham rats (4.8). Considering that at this time the castrated animals were not completely recuperated, these results could indicate an ongoing inflammatory immune response associated with Th1 activity in these animals. Also castrated animals developed antibodies to a diversity of MBP epitopes in comparison to sham rats, which presented a dominance of antibodies to MBP peptide p96–128. These results indicate that sex hormones levels regulate cell-mediated immunity and the specificity of anti-MBP antibodies related to the induction and development of acute EAE.     

Propentofylline and Iloprost Suppress the Production of TNF-αby Macrophages but Fail to Ameliorate Experimental Autoimmune Encephalomyelitis in Lewis Rats

Intracellular cAMP levels can be elevated by activation of cAMP-generating adenylate cyclase (AC) or inhibition of cAMP-cleavage by phosphodiesterases. Elevation of intracellular cAMP levels in immune cells inhibits production of some Th1-cytokines, particularly TNF-α, and results mainly in downregu-lation of the immune response. Experimental autoimmune encephalomyelitis (EAE) of Lewis rats is a disease mediated by type 1 T helper lymphocytes and macrophages and serves as a model of multiple sclerosis. In EAE we therefore tested the immunomodulatory potency of an AC-activating, stable prosta-cyclin analogue, iloprost, and of a potent and non-selective inhibitor of phosphodiesterases, propentofylline, which also has neuroprotective properties.Preventive treatment of Lewis rats with propentofylline (2×10 or 12.5 mg/kg/d), iloprost (2×10 or 12.5 μg/kg/d), or both did not significantly ameliorate clinical or histological signs of EAE actively induced by immunization with myelin basic protein (MBP) in complete Freund's adjuvant. Furthermore, adoptive transfer EAE (AT-EAE), passively induced by injection of encephalitogenic MBP-specific Th1 lymphocytes, was not altered in its course by the combined application of iloprost (2×10 μg/kg/d) and propentofylline (2×20 mg/kg/d) starting on the day of cell transfer. In vitro assays demonstrated that iloprost strongly and propentofylline moderately inhibited the production of TNF-α by macrophages and that iloprost in vivo similarly suppressed TNF-α secretion, although this effect was limited to a few hours after a single injection. In contrast to macrophages, TNF-α production by antigen-activated encephalitogenic T helper line cells in vitro was completely resistant to modulation by these agents. In addition, the presence of iloprost, propentofylline, or both drugs during activation of the line cells in vitro did not impair their encephalitogenicity in vivo.The findings delineate immunomodulatory effects of both substances, particularly of iloprost, but fail to support a possible therapeutic role of these agents in autoimmune inflammation of the central nervous system.     

Vasoactive Intestinal Polypeptide Suppressed Experimental Autoimmune Encephalomyelitis by Inhibiting T Helper 1 Responses

Vasoactive intestinal peptide (VIP) has been found to act as a potent anti-inflammatory factor through regulating the production of both anti- and pro-inflammatory mediators and promoting Th2-type responses. In this study, we used myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice to investigate the potential effects of VIP on multiple sclerosis. Our results showed that in vivo treatment of EAE-induced mice with VIP had great protective benefit at both clinical and histological levels. Disease suppression was associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and influencing the expression of pro-inflammatory cytokines including IFN-γ, IL-6 and IL-2 as well as chemotactic factors such as RANTES. In conclusion, the study provides evidence that VIP had great protective effect on EAE through its inhibition actions on pathogenic T cells and through a specific effect on the Th1 response.Haiyan Li and Yunhua Mei contributed equally to this work     

Inhibitory Effects of Incomplete Freund's Adjuvant on Experimental Autoimmune Encephalomyelitis

Freund's incomplete adjuvant (IFA), an aqueous/oil emulsion that is widely used in combination with antigenic proteins and peptides to induce tolerance, is considered to be immunologically inert. However, sporadic reports indicate that IFA may itself have inhibitory properties on induction of adjuvant induced arthritis and spontaneous diabetes. In the current study, the effects of IFA/saline were evaluated on the induction of experimental autoimmune encephalomyelitis (EAE) in three different strains of mice. IFA/saline given i.p. in two doses of >100 &#117 &#119 l 10 &#117 days apart were found to inhibit EAE induction to varying degrees in all three strains of mice in a dose dependent fashion. The IFA/saline injections inhibited both mitogen and antigen-induced T cell proliferation, induced elevated secretion of IFN- &#110 and IL-10 by neuroantigen specific T cells, and reduced expression of cytokines, chemokines, and chemokine receptors of CNS-infiltrating mononuclear cells. These data demonstrate for the first time a direct inhibitory effect of IFA/saline on EAE, and re-emphasize the need to properly control experiments using IFA to induce antigen-specific tolerance.     

Influence of Various Immunomodulators on the Induction of Experimental Autoimmune Encephalomyelitis in Lewis Rats

The effect of various immunomodulators on the induction of experimental autoimmune encephalomyelitis (EAE) is evaluated in the Lewis rat. Bordetella pertussis (BP) is the optimal inductor of EAE in this rat strain. Treatment of the animals with BP either before or after or simultaneously with guinea-pig spinal cord preparation (GpSC) resulted in an EAE about two weeks thereafter. Additional injection of living BCG, of CFA, IFA (incomplete Freund's adjuvant) or Vibrio cholerae neuraminidase (VCN) did not augment or mitigate the effect induced by BP or GpSC. Living BCG, IFA, VCN or Corynebacterium parvum (CP) did not induce EAE when given in combination with GpSC but without BP. CFA combined with GpSC only occasionally induced EAE. However, EAE could be induced by the combination of CFA and GpSC or IFA and GpSC in a part of the animals tested if they had been pretreated or simultaneously been injected with living BCG by intravenous route. EAE could not be enhanced by the additional injection of VCN. Surprisingly, most of the animals peracutely died after injection of CFA and BP in combination with GpSC when they had been pretreated with CP. This effect was most pronounced when pretreatment was done on day -4. No acute effect could be seen when CP was given simultaneously to CFA, BP and GpSC. Animals which did not peracutely succumb developed EAE similarly as those in the positive control groups. CP treatment simultaneously with BP but without CFA resulted in a reduction of the EAE specific mortality. This reduction could not be seen if treatment with CP was done after injection of GpSC and BP.     

Lingo‐1 Inhibited by RNA Interference Promotes Functional Recovery of Experimental Autoimmune Encephalomyelitis

Lingo‐1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo‐1 expression, and the impact of Lingo‐1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo‐1 short hairpin RNA (LV/Lingo‐1‐shRNA) were constructed to inhibit Lingo‐1 expression. LV/Lingo‐1‐shRNA of different titers were transferred into myelin oligodendrocyte glycoprotein‐induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo‐1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining.LV/Lingo‐1‐shRNA administration via ICV injection could efficiently down‐regulate the Lingo‐1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P < 0.01), especially in the 5 × 10⁸ TU/mL and 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups. The locomotor function score in the LV/Lingo‐1‐shRNA treated groups were significantly lower than the untreated or LVCON053 group from day 7 on. The 5 × 10⁸ TU/mL LV/Lingo‐1‐shRNA group achieved the best functional improvement (0.87 ± 0.11 vs. 3.05 ± 0.13, P < 0.001). Enhanced myelination/remyelination was observed in the 5 × 10⁷, 5 × 10⁸, 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups by LFB staining (P < 0.05, P < 0.01, and P < 0.05).The data showed that administering LV/Lingo‐1‐shRNA by ICV injection could efficiently knockdown Lingo‐1 expression in vivo, improve functional recovery and enhance myelination/remyelination. Antagonism of Lingo‐1 by RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE. Anat Rec, 297:2356–2363, 2014. © 2014 Wiley Periodicals, Inc.     

Enhanced Expression of Constitutive Endothelial Nitric Oxide Synthase by Astrocytes in the Spinal Cords of Rats with Experimental Autoimmune Encephalomyelitis

To investigate the expression of eNOS in a disease affecting the CNS, we induced EAE and analyzed the expression of eNOS by Western blot analysts and immunohistochemistry. Western blot analysis indicated that eNOS increased substantially in the spinal cords of rats with EAE compared with the spinal cords of normal and adjuvant sensitized rats. Immunohistochemistry revealed that eNOS was positive for some macrophages and astrocytes in EAE lesions. The vascular endothelial cells in EAE lesions also showed a light increase of eNOS immunoreactivity. This trial demonstrated that eNOS is increased in the diseased EAE spinal cord and that the source of eNOS is from the exogenous inflammatory cells and from the endogenous spinal cord cells including astrocytes.     

COX-2 Inhibitors Modulate IL-12 Signaling Through JAK-STAT Pathway Leading to Th1 Response in Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). IL-12 plays a crucial role in the pathogenesis of EAE/MS and inhibition of IL-12 production or IL-12 signaling was effective in preventing EAE. Cyclooxygenase (COX-2) is a key enzyme promoting inflammation in rheumatoid arthritis and tumor induced angiogenesis. Recent studies have shown that COX-2 inhibitors prevent EAE, however, their mechanism of action is not fully understood. In this study, we show that in vivo treatment (i.p.) with 100 μg COX-2 selective inhibitors (LM01, LM08, LM11, and NS398), on every other day from day 0 to 30, significantly reduced the incidence and severity of EAE in SJL/J and C57BL/6 mice. Further analyses showed that the COX-2 inhibitors reduced neural antigen-induced IL-12 production, T cell proliferation and Th1 differentiation ex vivo and in vitro. The COX-2 inhibitors also decreased IL-12-induced T cell responses through blocking tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 proteins in T cells. These results demonstrate that COX-2 inhibitors ameliorate EAE in association with the modulation of IL-12 signaling through JAK-STAT pathway leading to Th1 differentiation and suggest their use in the treatment of MS and other Th1 cell-mediated autoimmune diseases.     

Enhanced Expression of Constitutive Endothelial Nitric Oxide Synthase by Astrocytes in the Spinal Cords of Rats with Experimental Autoimmune Encephalomyelitis

To investigate the expression of eNOS in a disease affecting the CNS, we induced EAE and analyzed the expression of eNOS by Western blot analysts and immunohistochemistry. Western blot analysis indicated that eNOS increased substantially in the spinal cords of rats with EAE compared with the spinal cords of normal and adjuvant sensitized rats. Immunohistochemistry revealed that eNOS was positive for some macrophages and astrocytes in EAE lesions. The vascular endothelial cells in EAE lesions also showed a light increase of eNOS immunoreactivity. This trial demonstrated that eNOS is increased in the diseased EAE spinal cord and that the source of eNOS is from the exogenous inflammatory cells and from the endogenous spinal cord cells including astrocytes.