甲基强的松龙治疗实验性变态反应性脑脊髓炎的作用机制

目的:研究细胞因子、T细胞凋亡和淋巴细胞增殖在实验性变态反应性脑脊髓炎(EAE)形成中的作用及甲基强的松龙(MP)治疗EAE的作用机制。方法:采用人脑纯化的髓鞘碱性蛋白(MBP)与完全福氏佐剂免疫Lewis大鼠,建立EAE动物模型。用双抗体夹心ELISA法检测各组大鼠血清中IL-10、TNF-α、IFN-γ的含量:流式细胞仪检测外周血T细胞凋亡;3H-TdR释放法检测外周血淋巴细胞转化率。结果:与对照组比较,EAE组的外周血IFN-γ、TNF-α水平明显增高,IL-10水平明显降低,MP治疗后IFN-γ和TNF-α水平下降,IL-10浓度上调。MP还诱导外周血T细胞凋亡和抑制MBP致敏淋巴细胞增殖并呈剂量依赖性。结论:应用人MBP成功建立EAE大鼠模型,MP可能通过调节Th细胞因子格局、促进Th2细胞因子分泌、抑制MBP致敏淋巴细胞增殖及外周血T细胞凋亡而发挥治疗多发性硬化的作用。     

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4⁺ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73–84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73–84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73–84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73–84:1028—coimmunized protected rats proliferate and secrete interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in vitro in response to MBP73–84. Furthermore, coimmunized protected rats harbor a population of MBP73–84—reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73–84—reactive T cells or alteration of the cytokine secretion profile of the MBP73–84—reactive cell population. Rather, MBP73–84—reactive potentially encephalitogenic T cells are primed in these protected animals. © 1996 Wiley-Liss, Inc.     

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin,tumor necrosis factor α and tumor necrosis factor β

The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-β (=lymphotoxin-α) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-α peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-γ (IFN-γ) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-γ. The TNF-β mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-α was suggested from these observations that TNF-α mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.     

Synergistic interaction between Toll-like receptor agonists is required for induction of experimental autoimmune encephalomyelitis in Lewis rats

To investigate whether TLR agonists can replace mycobacteria in adjuvant to induce EAE in Lewis rats, we immunized rats with MBP peptide (MBP(68-86)) in IFA, supplemented with TLR agonists. Rats immunized with MBP(68-86) plus CpG-ODN or LPS in IFA did not develop EAE. In contrast, rats immunized with MBP(68-86) plus CpG-ODN and LPS in IFA developed clinical EAE. Spleen cells proliferated and secreted IFN-gamma in response to MBP(68-86), and secreted IL-6 and IL-12p40 in response to CpG-ODN and LPS. However, rats immunized with MBP(68-86) plus CpG-ODN and PolyI:C, a TLR3 agonist, did not develop EAE. We conclude that selected combinations of TLR agonists can facilitate the induction of EAE by MBP peptide via the innate immune system.     

特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值

目的特殊染色鉴定小剂量豚鼠脊髓匀浆诱导Wistar大鼠实验性自身免疫性脑脊髓炎模型的价值,并观察其病理的改变。方法按脊髓重量与冰盐水体积之比为1:5的比例制备豚鼠脊髓匀浆抗原,免疫Wistar大鼠,建立EAE模型;组织切片进行HE染色,三色染色,髓鞘碱性蛋白(MBP)及神经微丝(NF)免疫组化,光镜下观察病理改变。结果Wistar大鼠免疫后第16.07±4.25天发病,发病形式多样,除表现EAE经典症状外,尚出现痉挛状态、斜颈等特殊症状。HE染色发现神经组织内炎细胞浸润,血管"袖套"样病灶形成;三色染色可见轴突肿胀,呈串珠状,且不连续,着色不均匀;髓鞘结构层次紊乱,疏松,崩解;MBP及NF免疫组化研究发现病变组织内白质脱髓鞘及轴突损伤。结论三色染色结合MBP、NF免疫组化检测,较常规HE染色更能直接地、准确地显示EAE大鼠的病理变化,可推广应用。     

CD24 and myosin light polypeptide 2 are involved in prevention of experimental autoimmune encephalomyelitis by myelin basic protein-pulsed dendritic cells

We previously demonstrated that injection of myelin basic protein-pulsed (MBP-pulsed)--but not of unpulsed--autologous bone marrow-derived dendritic cells (DC) efficiently prevents experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To define the molecules involved, we used 3 groups of rats pretreated subcutaneously with MBP-DC, or unpulsed DC, or PBS (control EAE). Four weeks later, all rats were immunized with encephalitogenic MBP peptide and adjuvant. Microarray analyses were done to screen for genes that differ among the 3 groups. Based on microarray analysis data, we used real-time PCR to measure expression of six probably involved genes in draining lymph node cells obtained on day 0, day 7 and day 14 post immunization (p.i.). Two of these 6 genes were consistently altered in both microarray analyses and RT-PCR. They are CD24 antigen being persistently low, and myosin light polypeptide 2 (Myl2) being high in the acute immune response in MBP-DC pretreated rats that develop resistance to EAE. These two genes could be targeted to treat EAE and, possibly, multiple sclerosis.     

Similar pattern of MCP-1 expression in spinal cords and eyes of Lewis rats with experimental autoimmune encephalomyelitis associated anterior uveitis

Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family responsible for the recruitment of T cells that have been found during inflammation of the spinal cord in experimental autoimmune encephalomyelitis (EAE) in Lewis rats immunized with myelin basic protein (MBP). Lewis rats injected with MBP also developed anterior uveitis (AU), which coincided with the onset of EAE. In the present studies, we examined the expression and distribution of MCP-1 in the eye and spinal cord during disease and compared it to the expression of Th1 cell type cytokines. Initially, MCP-1 expression was detected at the preclinical phase in the iris/ciliary body and lumbar spinal cord and increased during the course of EAE/AU. Mononuclear infiltrating cells and endothelial cells and astrocytes of the CNS could be identified as a source of MCP-1 by in situ hybridization. Kinetics of expression of Th1 characteristic cytokines such as IL-2 and IFNγ was in agreement with the expression of MCP-1 chemokine. Moreover, induction of the gene expression of MCP-1 seemed to occur earlier than that of MIP-2, and it correlated with increasing disease severity. MCP-1 seems to contribute to the initial recruitment of inflammatory cells into both the tissues of the eye and CNS over the course of disease. J. Neurosci. Res. 50:531–538, 1997. © 1997 Wiley-Liss, Inc.     

Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors

The proinflammatory Th1 cytokine, tumor necrosis factor-α (TNFα), the cell death signaling molecule FasL, and several extracellular matrix degrading metalloproteinases have been implicated in the pathogenesis of multiple sclerosis (MS). The latter enzymes, as well as TNFα-converting enzyme and FasL-converting enzyme, can be blocked by matrix metalloproteinase inhibitors (MMPIs). In this study, we show that a potent MMPI was clinically effective in an animal model for MS, experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse. Efficacy was remarkable, as indicated by blocking and reversal of acute disease and reduced number of relapses and diminished mean cumulative disease score in chronic relapsing animals. Also, demyelination and glial scarring were significantly decreased in MMPI-treated mice with chronic relapsing EAE, as was central nervous system gene expression for TNFα and fasL. It is interesting that expression of the beneficial cytokine interleukin-4 (IL-4) was increased, and IL-4 was expressed on glial cells. The relevance of these compounds for MS was underscored by their ability to specifically inhibit TNFα shedding and cytotoxicity of myelin-autoreactive human cytotoxic CD4⁺ T-cell clones. This is the first report to show a positive effect by MMPIs on chronic relapsing EAE, its central nervous system cytokine profile, and on TNFα shedding by human myelin-autoreactive T cells.     

T and B cell responses to myelin basic protein and encephalitogenic epitopes

The major encephalitogenic epitope of myelin basic protein (MBP) for the Lewis rat includes residues 68–84, although a minor epitope has been localized to MBP residues 87–99. We synthesized MBP68–84 and MBP87–99, and immunized rats with these peptides or with MBP in complete Freund's adjuvant (CFA). MBP and MBP68–84 induced paralytic experimental autoimmune encephalomyelitis (EAE) at equimolar concentrations, whereas significantly higher dosages of MBP87–99 were required to elicit paralytic disease. Spleen cells (SpC) from MBP- or MBP68–84-immunized rats could be activated with either MBP or MBP68–84 to transfer EAE to recipients. Anti-MBP antibodies were detected by ELISA in rats immunized with MBP-CFA, and anti-MBP68–84 specific antibodies were present in serum obtained from MBP68–84-immunized animals. However, these antibodies were non-cross reactive. MBP87–99 elicited only a meager antibody response to the immunizing peptide, and cross reactivity with MBP was not observed. Thus, although MBP and each peptide exhibited encephalitogenic activity, and MBP and MBP68–84 were cross reactive at the T cell level, the absence of cross reactivity at the humoral level indicates that significant immunological differences exist between MBP and the synthetic determinants, which may reflect differences in epitope recognition by T and B lymphocytes.     

Heat shock proteins and experimental autoimmune encephalomyelitis (EAE): I. Immunization with a peptide of the myelin protein 2′,3′ cyclic nucleotide 3′ phosphodiesterase that is cross-reactive with a heat shock protein alters the course of EAE

We describe sequence similarity and immunologic cross-reactivity between a peptide of the mycobacterial hsp, HSP65, and the myelin protein 2′,3′ cyclic nucleotide 3′ phosphodiesterase (CNP). We demonstrate that immunization with the homologous cross-reactive CNP peptide (hsp-CNP peptide) has significant biological consequences. Rats immunized with hsp-CNP peptide in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) produce large amounts of peptide-specific antibody. Isotypes of antibodies in animals immunized with peptide in CFA are IgG1 and IgG2a. Isotypes of antibodies in rats immunized with peptide in IFA are predominantly IgG1, with low titers of IgG2a. T cell proliferative responses to HSP65 are present in rats immunized with peptide in CFA. T cell responses to HSP65 initially are absent in rats immunized with peptide in IFA but develop over time. T cell proliferative responses to hsp-CNP peptide were not detected. None of the groups of rats developed clinical or histologic evidence of experimental autoimmune encephalomyelitis (EAE). To induce EAE, rats preimmunized with hsp-CNP peptide were challenged with guinea pig spinal cord (GPSC) emulsified in CFA. Rats preimmunized with peptide in CFA developed severe EAE. Rats preimmunized with hsp-CNP peptide in IFA were protected from EAE, with both a lower incidence and severity of disease. Injecting the murine monoclonal antibody recognizing the shared HSP65 and CNP epitope did not protect against EAE. Our data suggest that a Th2 pattern of immune response to a CNP peptide that itself is non-encephalitogenic protects against EAE. Immune responses to either hsp or myelin proteins cross-reactive with hsp may play an important role in the development of EAE. © 1996 Wiley-Liss, Inc.     

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.     

Cloning of myelin basic protein-reactive T cells from the experimental allergic encephalomyelitis-resistant rat strain, LER

Rats of the LER inbred strain are resistant to the active induction of experimental allergic encephalomyelitis (EAE), although they are susceptible to adoptively transferred EAE when they are injected with encephalitogenic T cells from EAE-susceptible Lewis rats. The mechanism of resistance remains to be elucidated. We report here that myelin basic protein (MBP)-specific T cells can be cloned from LER rats immunized with MBP, that these CD4⁺ LER T cells can recognize the encephalitogenic peptide (MBP-EP) and will divide vigorously when it is presented to them, and that these T cells bear Vβ8 + TCR chains. Nevertheless, in contrast to Lewis T cells with the same specificity and TCR β chains, LER T cells from MBP-EP-specific clones cannot induce EAE when adoptively transferred into naive rats of either strain. Thus, LER T cells can assemble and use a TCR with the canonical encephalitogenic Vβ8.2-Dβ-Jβ region in response to immunization with MBP, yet they continue to display resistance to EAE.     

T cell immunity and interferon-γ secretion during experimental allergic encephalomyelitis in Lewis rats

An immunospot assay that detects single secretory cells was used to enumerate interferon-γ secreting cells (IFN-γ-sc) in mononuclear cell suspensions from the central nervous system (CNS) and peripheral lymphoid organs after actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. In the CNS compartment there was a significant increase in the number of IFN-γ-sc preceding the onset of the clinical signs of EAE. Both in rats with EAE and rats immunized with Freund's complete adjuvant (FCA) the number of IFN-γ-sc increased in peripheral lymphoid organs, as compared to non-immunized controls. In view of the potent immunoregulatory effects of IFN-γ, its intra-CNS secretion may play a crucial role for clinicophatological events in EAE.To study the numbers of primed T cells that in response to myelin antigens produced IFN-γ, mononuclear cell suspensions from peripheral lymphoid organs were precultured to allow for antigen uptake, presentation and T cell triggering, followed by enumeration of IFN-γ-sc. T cells responding to a peptide of myelin basic protein (MBP) that previously have been shown encephalitogenic in Lewis rats, appeared initially and were quantitatively dominant over the course of EAE. Later, T cell reactivities to multiple regions of MBP appeared, showing that the concept of immunodominance in EAE is non-absolute and time dependent.Splenocyte cultures from EAE rats exposed to the different antigens showed a reduced number of IFN-γ-sc compared to cultures not exposed to antigen, suggesting an antigwn-induced suppression of T cell effector molecules.     

Effect of timing of intravenous administration of myelin basic protein on the induction of tolerance in experimental allergic encephalomyelitis

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 (na?ve 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 na?ve 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 na?ve 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 na?ve 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.     

Studies on the refractoriness to reinduction of experimental allergic encephalomyelitis in Lewis rats that have recovered from one episode of the disease.

Lewis rats immunized with myelin basic protein (MBP) in Freund's complete adjuvant (FCA) suffer from a single episode of paralysis from which they recover spontaneously and become refractory to induction of further episodes of paralysis by reimmunization with MBP in FCA. We show that there is a transient period immediately after recovery when a second episode of paralysis can be induced in some animals by reimmunization with MBP in FCA, or soluble MBP prior to the development of complete refractoriness. We also show that rats that are refractory to the induction of experimental allergic encephalomyelitis (EAE) by active immunization with MBP in FCA are also refractory to the adoptive transfer of EAE by in vitro-activated MBP-primed lymphocytes.     

Suppression of experimental autoimmune myasthenia gravis and experimental allergic encephalomyelitis by oral administration of acetylcholine receptor and myelin basic protein: double tolerance

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChR or MBP and complete Freund's adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChR + MBP + CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-γ secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-γ in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR + MBP + CFA alleviated clinical disease as well as AChR- and MBP-specific B cell responses and autoantigen-induced IFN-γ secretion and production, but upregulated antigen-induced TGF-β mRNA expression in lymph node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-γ and augmentation of TGF-β are pivotal in tolerance induction.     

Importance of cryptic myelin basic protein epitopes in the pathogenicity of acute and recurrent anterior uveitis associated with EAE

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which can relapse without recurring of EAE. In this study, we analyzed the repertoire of MBP epitopes that play a role in acute and recurrent AU by injection of MBP synthetic peptides. In addition to the encephalitogenic epitopes 69-89 and 87-99, several cryptic epitopes were found to be strongly uveitogenic in Lewis rats upon immunization with synthetic peptides, including 100-120, 121-140 and 142-167. However, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. Immunization with intact MBP was not essential for the induction of the recurrence of AU. The responses of T cells from lymph nodes and spleens showed a dominant response to the original disease-induced epitope with responses to secondary epitopes. In conclusion, the analysis of pathogenic determinants important for the induction of uveitis provides further evidence that MBP-specific T cells also contribute to the pathogenesis of anterior uveitis. Moreover, this also suggests that a distinct immunoregulatory mechanism exists in the eye and spinal cord because of the uniqueness of the epitope 1-20 in AU but not EAE, and the capability of MBP-specific T cells of inducing AU without EAE.     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.