经鼻黏膜给予MBP68-86和87-99协同免疫预防Lewis大鼠EAE的实验研究

目的探讨鼻黏膜给予MBP68-86和87-99协同免疫预防Lewis大鼠实验性自身免疫性脑脊髓炎(EAE)的作用。方法合成3条不同的碱性髓鞘蛋白(MBP)多肽(MBP68-86、87-99和非致脑炎性肽段110-128),在用豚鼠MBP(gp-MBP)加弗氏完全佐剂免疫Lewis大鼠前的11、10、9、8和7d,经鼻黏膜分别给予MBP多肽,观察其对EAE的保护作用。结果致脑炎性肽段MBP68-86和87-99都有保护作用,其中MBP68-86的保护作用更强;而MBP110-128没有保护作用。鼻黏膜给予MBP68-86 87-99的混合物,在相对低的剂量可完全阻断gp-MBP引发的EAE。淋巴细胞增殖实验和IFN-γELISPOT检测显示,与鼻黏膜给予大鼠MBP110-128组相比,鼻黏膜给予大鼠MBP68-86 87-99可降低T细胞对于MBP的反应性,淋巴结单核细胞中表达IFN-γ和TNF-αmRNA的细胞数减少,而两组表达TGF-β及IL-4mRNA的淋巴细胞数都低。结论鼻黏膜给予致脑炎性MBP多肽能够导致抗原特异性T细胞耐受,对gp-MBP引发的EAE提供不完全的保护,MBP68-86和MBP87-99具有协同作用。鼻黏膜给予多肽引发的免疫耐受与非调节机制有关。     

Combined nasal administration of encephalitogenic myelin basic protein peptide 68-86 and IL-10 suppressed incipient experimental allergic encephalomyelitis in Lewis rats

Mucosal administration of low doses of myelin basic protein (MBP) peptide 68-86 (MBP 68-86) or anti-inflammatory cytokine IL-10 effectively prevented experimental allergic encephalomyelitis (EAE), but failed to suppress the disease if given after 7 days postimmunization (p.i.), i.e., after T cell priming had occurred. We anticipated that combined administration of autoantigen and IL-10 can treat incipient EAE. Lewis rats with EAE actively induced with MBP 68-86 and complete Freund's adjuvant received 120 microg MBP 68-86 + 200 ng IL-10 per rat per day from day 7 p.i. and for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss, and shorter duration of EAE than rats receiving MBP 68-86 or IL-10 only or PBS. EAE amelioration was associated with decreased infiltration of ED1(+) macrophages and CD4(+) T cells within the central nervous system and with decreased proliferative responses of lymph node cells, indicating that combined administration of MBP 68-86 and IL-10 induced immune hyporesponsiveness. IFN-gamma secretion as well as IFN-gamma, TNF-alpha, IL-4, and IL-10 mRNA expression by lymph node MNC was down-regulated in the treated rats. Immune hyporesponsiveness, rather than immune deviation or regulatory mechanisms, seems to be responsible for the protection of EAE after autoantigen + IL-10 administration by the nasal route.     

Inhibition of experimental autoimmune encephalomyelitis in SJL mice by oral administration of retro-inverso derivative of encephalitogenic epitope P87-99.

Retro-inverso modification of peptides preserves parent peptide overall topology and provides at the same time stability to proteolysis, leading to derivatives with prolonged half-life in vitro and in vivo. In this study the encephalitogenic epitope P87 - 99 of myelin basic protein has been prepared in the retro-inverso form to examine its biological activity in a murine model of multiple sclerosis. Experiments of in vivo T cell tolerance induction in SJL mice revealed that the retro-inverso peptide was able to induce a selective T cell hyporesponsiveness, as measured by a reduction in the proliferative response of lymphnode T cells after antigen challenge. Oral administration of retro-inverso peptide decreased the disease severity significantly and delayed considerably the disease onset in treated mice. Enhancement of resistance to proteolysis by retro-inverso modification of encephalitogenic epitopes may increase the therapeutic value of oral tolerance induction in the treatment of multiple sclerosis and other Th1-associated inflammatory disorders.     

Suppression of ongoing experimental allergic encephalomyelitis (EAE) in Lewis rats: synergistic effects of myelin basic protein (MBP) peptide 68-86 and IL-4

Mucosal myelin autoantigen administration effectively prevented EAE, but mostly failed to treat ongoing EAE. Patients with multiple sclerosis (MS), for which EAE is considered an animal model, did not benefit from oral treatment with bovine myelin. We anticipated that autoantigen, administered together with a cytokine that counteracts Th1 cell responses, might ameliorate Th1-driven autoimmune disease, and that nasal administration might considerably reduce the amounts of antigen + cytokine needed for treatment purposes. Lewis rats with EAE actively induced with myelin basic protein peptide (MBP 68-86) and Freund's complete adjuvant (FCA), received from day 7 post-immunization, i.e. after T cell priming had occurred, 120 microg MBP 68-86 + 100 ng IL-4 per rat per day for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss and shorter EAE duration compared with rats receiving MBP 68-86 or IL-4 only, or PBS. EAE amelioration was associated with decreased infiltration of ED1+ macrophages and CD4+ T cells within the central nervous system, and with decreased interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and enhanced IL-4, IL-10 and transforming growth factor-beta (TGF-beta) responses by lymph node cells. Simultaneous administration of encephalitogenic peptide + IL-4 by the nasal route thus suppressed ongoing EAE and induced IL-4, IL-10 and TGF-beta-related regulatory elements.     

Inhibition of entire myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats by major histocompatibility complex class II-binding competitor peptides

Previous studies have shown that major histocompatibility complex (MHC) blockade by competitor peptides with high MHC class II binding affinity can prevent peptide-induced experimental autoimmune encephalomyelitis (EAE). However, none of these studies addressed the question whether this approach could also be used to prevent EAE induced with a multivalent antigen. In this report we show the effect of competitor peptides co-immunized during EAE induction with entire guinea pig myelin basic protein (MBP) in Lewis rats. As MHC class II binding competitor peptides we used one nonimmunogenic disease-nonrelated peptide, and two immunogenic peptides, one EAE-related and one non-EAE-related. The respective efficacy of these three competitor peptides to inhibit MBP-induced proliferation of an encephalitogenic T cell line in vitro correlated with their respective MHC binding affinity. Co-immunization of the competitor peptides during disease induction with entire MBP resulted in a competitor concentration-dependent inhibition of clinical signs of EAE. These results demonstrate that, although polyclonal T cell responses to MBP were not completely inhibited, co-administration of immunogenic or nonimmunogenic either EAE-related or non-EAE-related MHC class II binding competitor peptides can inhibit the development of EAE induced with entire MBP.     

鼻黏膜诱导免疫耐受预防实验性自身免疫性脑脊髓炎机制的研究

目的：探讨鼻黏膜免疫耐受对实验性自身免疫性脑脊髓炎（EAE）的预防机制以及与耐受原相关的剂量依赖性。方法：建立Lewis大鼠EAE模型，用不同种属和剂量髓鞘碱性蛋白（MBP）诱导免疫耐受，评估EAE临床发病情况；检测特异性淋巴细胞增殖反应ELISPOT检测特异性单个核细胞IFN-γ表达及原位杂交方法检测相关细胞因子的变化。结果：经鼻黏膜给予低剂量（30μg／rat）和高剂量（600μg／rat）特异性抗原可诱导免疫耐受，抑制EAE的发生。淋巴细胞增殖实验显示，高、低剂量耐受组与对照组相比均可抑制特异性淋巴细胞增殖反应（P〈0．001；P〈0．01）。ELISPOT结果表明，与对照组相比高、低剂量耐受组单个核细胞IFN-γ表达数量明显降低（P〈0．01；P〈0．001）。原位杂交结果显示，与对照组相比，低剂量组IL-4mRNA的表达水平明显增高（P〈0．001），高剂量组未见明显增高（P〉0．05）。结论：经鼻黏膜可诱导机体产生免疫耐受，预防EAE的发生。给予低剂量抗原可引起Th1／Th2分泌的细胞因子发生偏离，产生免疫耐受。     

不同磷脂肽段诱导的实验性自身免疫性脑脊髓炎模型病理特征多样性的研究

目的:探索是否不同磷脂肽段可以诱导Lewis大鼠产生不同的病理学表现。方法:采用髓鞘碱性蛋白82-99(MBP82-99)、MBP68-86、髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫Lewis大鼠,每天进行神经功能评分。免疫后取各组大鼠大脑、小脑、脑干和脊髓组织,观察炎性细胞浸润部位和浸润程度、有无脱髓鞘和轴索损害。结果:MBP68-86和MBP82-99两组大鼠的大脑、脑干、小脑和脊髓组织均有炎性细胞浸润,脊髓炎症程度重于其他部位;MBP68-86和MBP82-99诱导的脊髓炎症程度无统计学差异;MOG35-55组大鼠仅脊髓组织受累,且炎症程度明显低于MBP组,其余各组未见炎性细胞浸润。各组大鼠神经组织均未见脱髓鞘和轴索受累。结论:不同磷脂肽段诱导Lewis大鼠神经组织炎性细胞浸润的分布和程度不同。     

Dose-dependent mechanisms relate to nasal tolerance induction and protection against experimental autoimmune encephalomyelitis in Lewis rats.

Nasal administration of soluble antigens is an exciting means of specifically down-regulating pathogenic T-cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T-cell-mediated animal model for human multiple sclerosis. Nasal administration of guinea-pig (gp)-MBP at a dose as low as 30 micrograms/rat can completely prevent gp-MBP-induced EAE, whereas nasal administration of bovine (b)-MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T-cell proliferation and interferon-gamma (IFN-gamma)-ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 micrograms/rat) of gp-MBP, but not after administration of b-MBP. Rats tolerized with both doses of gp-MBP had also abrogated MBP-induced IFN-gamma mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate-buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp-MBP transferred protection to actively induced EAE. Low-dose (30 micrograms/rat) gp-MBP-tolerized rats also had high numbers of interleukin-4 (IL-4) mRNA-expressing lymph node cells, while high-dose (600 micrograms/rat) gp-MBP-tolerized rats had low numbers of IL-4 mRNA-expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose-dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.     

Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2

The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab.     

Prevention of experimental autoimmune encephalomyelitis by encephalitogenic epitope sequence simplified derivatives

The encephalitogenic epitope P81-100 from mouse myelin basic protein was used to generate two simplified derivatives with glycine substitutions in alternating positions which were tested for their biological activity in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis. While both derivatives were unable to induce in mice the disease at the same parent peptide P81-100 dosage, T cell proliferation assays demonstrated their ability to compete with the parental peptide in a dose related manner. Experiments of cell surface binding and T cell tolerance revealed a different behavior of the two derivatives, suggesting different roles in the MHC blockade or T cell tolerance. On induction of encephalomyelitis in animals by P81-100 treatment, one variant proved in vivo to be very effective in protecting from the disease.     

Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in Lewis rats.

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) which causes paralysis. Several studies have reported the involvement of Ia antigen-expressing cells in the pathogenesis of EAE. Interferon-gamma (IFN-gamma) can induce Ia antigen expression on a wide range of cells. We examined the effect of IFN-gamma on EAE in Lewis rats. Systemically administered IFN-gamma did not change the disease course of EAE, whereas IFN-gamma applied locally into the ventricular system of the CNS resulted in complete suppression of clinical signs. Furthermore, we found that systemic administration of anti-IFN-gamma just prior to the onset of clinical symptoms resulted in a more severe disease course. We conclude that IFN-gamma is capable of exerting a suppressive action in EAE, possibly through induction of Ia antigen expression or through the induction of suppressive mechanisms locally in the CNS.     

Inhibition of experimental autoimmune uveoretinitis by oral administration of S-antigen and synthetic peptides.

S-Antigen, a photoreceptor cell protein, is highly efficient in inducing experimental autoimmune uveoretinitis (EAU), a severe inflammation of the uveal tract and retina of the eye. S-Antigen and six synthetic peptides, all of which correspond to known T-cell or B-cell recognition sites, were tested for their ability to induce oral tolerance to EAU in LEW rats. Feeding three 1-mg doses of native S-Antigen or three doses of one synthetic peptide, designated BSA(343-362) (200 micrograms per dose), reduced the incidence and severity of EAU induced by immunization with 50 micrograms of S-Antigen. Another peptide, BSA(270-289), was able to inhibit EAU only when a low dose (10 micrograms) of the uveitogenic S-Antigen was used to induce EAU. Animals which received 200 micrograms doses of four other immunologically active peptides, BSA(31-51), BSA(143-162), BSA(303-327) and BSA(333-352), were not significantly protected. Furthermore, animals fed BSA(343-362) were significantly less susceptible to EAU induced by adoptive transfer (tEAU) of the uveitogenic R9 T-cell lines. Con A-activated lymphocytes purified from spleens of rats fed peptide BSA(343-362) transferred partial resistance to tEAU induced by adoptive transfer of R9 line cells. The resistance of orally tolerized rats to induction of EAU by adoptive transfer of an activated, pathogenic T-cell line, and the ability of lymphocytes from orally-tolerized animals to transfer resistance to tEAU shows that effector mechanisms can be inhibited by oral feeding as well as the afferent mechanisms reported here and elsewhere. Circulating levels of IgG specific for S-Antigen were not affected by feeding any of the peptides.     

Peptide T does not ameliorate experimental autoimmune encephalomyelitis (EAE) in Lewis rats

Peptide T has been shown to inhibit T cell activation and cytokine production and function. Moreover, it has been reported to be a safe treatment in humans. We have studied the ability of peptide T to prevent or ameliorate EAE in Lewis rats. Peptide T was administered subcutaneously at different doses and phases of the disease according to several treatment protocols, but we could not observe a consistent effect of peptide T ameliorating the disease. Lymph node cell proliferation and IL-4 and interferon-gamma production were also studied. We conclude that peptide T neither prevents nor ameliorates EAE in Lewis rats.     

电针治疗对实验性自身免疫性脑脊髓炎大鼠体内产生细胞因子的影响

目的 观察电针刺激足三里穴位对实验性自身免疫性脑脊髓炎(EAE)大鼠体内分泌的细胞因子的影响.方法 将免疫大鼠随机分为四组:用完全弗氏佐剂免疫组(CFA组);用MBPB68-86B多肽与完全弗氏佐剂免疫组(EAE组);用MBPB68-86B多肽免疫后的大鼠电针刺激足三里穴位组和用MBPB68-86B多肽免疫后的大鼠电针刺激非穴位组.分别于免疫后第7天、14天和21天采用ELISA方法检测大鼠血浆中IFN-γ、IL-4、IL-17、IL-6和TGF-β细胞因子水平.结果 EA组与EAE组大鼠血浆中IL-4水平有所升高(第14天P=0.027,第21天P=0.36);IFN-γ表达量在第14天EA组明显低于EAE组(P=0.00021);IL-6在三个时间点EA组均明显低于EAE组(P=0.0078,P=0.00047,P=0.00093);IL-17免疫21天,EA组明显低于EAE组(P=0.0012);TGF-β在第7天和第14天EA组明显高于EAE组(P=0.020,P=0.041)结论 电针刺激足三里穴位对EAE大鼠的治疗作用,通过降低炎症细胞因子的产生,促进抑炎细胞因子的产生而发挥作用.     

Experimental autoimmune encephalomyelitis in hybrids between Lewis and Brown Norway rats

The susceptibility to experimental autoimmune encephalomyelitis (EAE) in Lewis (Lew) and Brown Norway (BN) rats was studied in breeding experiments, evaluating EAE from clinical signs of the disease. The Lew strain is highly susceptible, the BN strain is resistant to EAE. F₁ hybrids between the strains show an intermediate susceptibility as described by earlier authors. Back-cross experiments verify that susceptibility is inherited in a complex way, at least according to a two-gene model previously suggested. Analysis of the F₁ hybrids showed a bi-modal distribution of clinical scores, one group of rats which appear to have the same degree of susceptibility as the Lew strain, and another group with very low susceptibility. Study of F₂ rats produced by F₁ rats with high or low susceptibility showed that this property was probably not inherited, arguing against a residual heterozygosity in the parental strains. As an alternative hypothetical explanation, the possibility of allogeneic exclusion of genes regulating suppression of EAE is discussed.     

Increased phosphorylation of caveolin-1 in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis

The expression of phospho-specific caveolin-1 (p-caveolin-1) was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that p-caveolin-1 was constitutively expressed in normal spinal cords and that it significantly increased in the spinal cord with EAE at both early and peak stages of EAE (P<0.05), and decreased slightly at the recovery stage of EAE. Immunohistochemistry showed that p-caveolin-1 was constitutively expressed in few vascular endothelial cells and glial cells in the spinal cords of normal rats. In EAE lesions, p-caveolin-1 was intensely immunostained in inflammatory T cells and macrophages. Therefore, we postulate that phosphorylation of caveolin-1 occurred in the inflammatory cells of EAE lesions, and that caveolin-associated cell activation is mainly associated with inflammatory cells that appear during early and peak stages of EAE.     

Intratracheal administration to the lung enhances therapeutic benefit of an MBP peptide in the treatment of murine experimental autoimmune encephalomyelitis

The treatment of autoimmune diseases by targeted down-regulation of autoantigen-specific cells has been accomplished by the administration of high doses of autoantigen. We performed direct comparisons between injection of myelin basic protein peptide and administration by several nonparenteral routes to determine whether route impacted benefit in the treatment of murine allergic encephalomyelitis, a model for multiple sclerosis. The range of effective peptide doses spanned over 1000-fold, and route of delivery played a major role in determining optimal dose. The oral route of administration was the least effective, requiring at least 50- to 100-fold more antigen than subcutaneous injection, which in turn required at least 10-fold more antigen than delivery of peptide to the lung using an intratracheal instillation. Intratracheal delivery was also considerably more effective than inhalation of peptide, and, unlike inhalation, resulted in obvious penetration of delivered material deep into the lung. The increase in therapeutic efficacy did not appear to result from slower systemic delivery of antigen. Accumulation of peptide on antigen presenting cells in the spleen and in the brain was less efficient using the intratracheal route of administration compared to subcutaneous injection, implicating a special role for the lung microenvironment in the induction of immune nonresponsiveness.     

Recovery mechanisms from experimental allergic encephalomyelitis in rats: analyses by using encephalitogenic T cell line

The recovery mechanism of acute experimental allergic encephalomyelitis (EAE) in Lewis rats was studied by using an encephalitogenic T cell line specific for myelin basic protein. Antigen-activated line cells were highly encephalitogenic, but unstimulated line cells were not encephalitogenic. The activated line cells returned to the unstimulated state in a few days in culture medium without antigen. This decline of proliferative and encephalitogenic activities of the activated line cells was also observed even if the activated line cells were continuously stimulated with the antigen. In addition, rats during the convalescent stage from acute EAE showed only mild clinical signs of EAE even by transfer of almost a lethal dose of activated line cells. Thus, self-limiting capacity of autoaggressive cells and attenuation of effector cell function during the convalescent stage seem to be involved in the recovery mechanism of EAE.     

Inhibition of experimental allergic encephalomyelitis by carrier administered prior to challenge with encephalitogenic peptide-carrier conjugate

Guinea pigs can be rendered unresponsive to experimental allergic encephalomyelitis (EAE) challenge by prior injections of myelin basic protein (BP) or peptides derived from BP. The synthesized tryptophan-containing peptide (TrpP, corresponding to residues 114-122) which is the primary encephalitogenic sequence in BP (for guinea pigs) had been conjugated to the non-central nervous system protein bovine serum albumin (BSA) and used for EAE challenge. It has been shown in this study that prechallenge treatment with the BSA carrier alone can significantly inhibit the induction of EAE by TrpP-BSA conjugate. That carrier treatment is immunologically specific was determined when prior treatment with cytochrome c did not protect animals from EAE challenge with TrpP conjugated to BSA. In addition, in disease induced with BP or TrpP, and while TrpP did protect guinea pigs challenged with TrpP, it did not protect those challenged with BP. This last finding raises the possibility of a second encephalitogenic sequence in BP or an important role for the carrier in the TrpP conjugate.