实验性自身免疫性脑脊髓炎大鼠血CD4+CD25+T细胞的研究

目的探讨实验性自身免疫性脑脊髓炎(EAE)动物模型血CD4 CD25 T细胞的变化及其意义。方法以豚鼠全脊髓匀浆(GPSCH)为抗原免疫Wistar大鼠,建立EAE的动物模型,采用三色流式细胞仪检测EAE和正常大鼠外周血CD4 CD25 T细胞的细胞数并进行比较;通过观察大鼠行为学及脑和脊髓的病理改变确定EAE。结果EAE模型大鼠的成功率为48.9%,EAE大鼠外周血CD4 CD25 T淋巴细胞数(5.29±4.00)显著低于正常对照组(12.61±2.24)(P<0.01)。结论EAE大鼠血CD4 CD25 T细胞数明显减少,CD4 CD25 T淋巴细胞对神经系统脱髓鞘疾病是一种保护因子。     

Control of experimental autoimmune encephalomyelitis by CD4+ suppressor T cells: peripheral versus in situ immunoregulation

The pathogenesis of experimental autoimmune encephalomyelitis (EAE) can be efficiently kept under control by specialized subsets of CD4+ T lymphocytes able to negatively regulate the function of T cells with encephalitogenic potential. A number of observations support a role for such suppressor T cells in controlling early phases of disease development at the level of peripheral lymphoid organs but there is also evidence suggesting immunoregulation within the central nervous system (CNS) microenvironment itself. This review evaluates the sites of regulation based on available data from distinct experimental models. We then discuss these aspects with reference to suppressor CD4+ T cells induced through the epicutaneous application of pure CNS antigens that confer long term protection against EAE. Finally, we give an overview of genes recently discovered to be important in regulation of the immune system that may also prove to be key players in the modulation of EAE and MS.     

Preferential increase of IL-2R+ CD4+ T cells and CD45RB- CD4+ T cells in the central nervous system in experimental allergic encephalomyelitis.

We examined lymphocytes isolated from the spinal cord (SC), peripheral blood (PB) and lymph nodes (LN) draining the immunization site of Lewis rats with acute experimental allergic encephalomyelitis (EAE). Cells were analysed for T cell subset markers CD4 (mAb W3/25) and CD8 (mAb OX8), for IL-2R (mAb OX39), and for high molecular mass leukocyte common antigen (LCA, CD45RB) expression (mAb OX22). T cells expressing high (CD45RB+) or low (CD45RB-) molecular mass LCA are of different maturational stages and/or separate lineages. CD4+ T cells were more predominant in SC than in PB and LN; CD8+ T cells were scarce in SC but common in PB and LN. Activated CD4+ T cells (IL-2R+) were common in the SC and LN but infrequent in blood. CD4+ T cells that were CD45RB+ were scarce in the SC. In contrast, the majority of CD4+ T cells in the PB and LN were CD45RB+. The preferential accumulation of IL-2R+ CD4+ T cells and of CD45RB- CD4+ T cells in the central nervous system (CNS) indicates that a selective mechanism directs cell egress into CNS lesions in EAE.     

Protection against experimental autoimmune encephalomyelitis requires both CD4 T suppressor cells and myelin basic protein-primed B cells

Suppressor cells that regulate experimental autoimmune encephalomyelitis (EAE) are present in rats that recover from the disease and can protect against the development of active EAE when transferred to normal recipients. Both CD4+ T suppressor cells, known to regulate EAE effector cell lymphokine production, and myelin basic protein (MBP)-primed B cells are required to transfer protection against EAE to normal recipients. Neither CD4+ T suppressor cells nor MBP-primed B cells alone could transfer protection. Moreover, the co-transfer of normal B cells with CD4+ T suppressor cells did not provide protection against EAE. These results suggest that the regulation of EAE and perhaps the recovery from acute clinical disease requires the interaction of two specific subpopulations of regulatory lymphocytes.     

雷公藤内酯醇对实验性自身免疫性脑脊髓炎外周血及中枢神经系统中CD4+T淋巴细胞的影响

目的 探讨雷公藤内酯醇（Tri）对实验性自身免疫性脑脊髓炎（EAE）外周血及中枢神经系统中CD4^＋T淋巴细胞的影响，为Tri临床应用提供实验依据。方法 建立EAE大鼠模型，给予Tri预防性治疗．流式细胞术检测大鼠外周血的CD4^＋T淋巴细胞变化，免疫组化观察中枢神经系统中CD4^＋T淋巴细胞的浸润情况。结果 与生理盐水模型组比较，Tri治疗组的发病率显著降低，中枢神经系统中CD4^＋T淋巴细胞的浸润显著减少，而外周血CD4^＋T淋巴细胞的量变化不大。结论 Tri可能通过抑制CD4^＋T淋巴细胞在中枢神经系统的浸润，而阻止EAE的发病，提示Tri在治疗人类多发性硬化方面有一定应用前景。     

High cell surface expression of CD4 allows distinction of CD4(+)CD25(+) antigen-specific effector T cells from CD4(+)CD25(+) regulatory T cells in murine experimental autoimmune encephalomyelitis

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up-regulate cell surface expression of CD4. The CD4(high) sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25(+) sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses.     

载脂蛋白E拟肽对实验性自身免疫性脑脊髓炎小鼠脑脊髓CD4+、CD8+T淋巴细胞表达的影响

目的 探讨载脂蛋白(Apo)E拟肽对实验性自身免疫性脑脊髓炎(EAE)小鼠脑脊髓CD4+、CD8+T淋巴细胞表达的影响.方法 40只C57BL/6J雌性小鼠随机分成EAE组、EAE治疗组、正常对照组、正常治疗组;采用髓鞘少突胶质细胞糖蛋白制备的完全抗原诱导EAE模型小鼠.免疫诱导次日,EAE治疗组和正常治疗组小鼠每隔2d皮下注射ApoE拟肽,EAE组和正常对照组小鼠皮下注射等量的生理盐水.免疫诱导后各组每日进行神经功能缺损评分(NDS);35 d后用免疫组化检测各组小鼠脑脊髓CD4+T细胞、CD8+T细胞的表达.结果 EAE治疗组NDS的峰值及终末评分显著低于EAE组(均P＜0.05).与正常对照组及正常治疗组比较,EAE组大脑、脑干和脊髓中CD4+T细胞数明显增高,大脑CD8+T细胞数明显增高(均P＜0.05).EAE治疗组小鼠大脑、脑干、脊髓组织CD4+T细胞表达显著低于EAE组(均P＜0.05);两组间CD8+T细胞表达水平的差异无统计学意义.结论 ApoE拟肽可抑制CD4+T细胞的表达,减轻免疫炎症反应,对EAE小鼠有神经保护作用;而对CD8+T细胞的表达无明显影响.     

Loss rather than downregulation of CD4 T cells as a mechanism for remission from experimental allergic encephalomyelitis

SJL/J mice recover from clinical signs of experimental allergic encephalomyelitis (EAE) 2 to 3 days following the onset of the initial attack. The immunoregulatory events that induce clinical recovery are not well understood. In this paper we have compared the activation state of the T cells infiltrating the central nervous system (CNS) in symptomatic and remitted mice. We isolated mononuclear cells from the CNS at various time points during the course of EAE and used flow cytometry to describe the kinetics of CNS infiltration by CD45⁺, CD2⁺, CD3⁺, TCRαβ⁺, CD4⁺ cells. There was a 30-fold reduction in the number of CNS CD4⁺ T cells in remitted mice 10 days following the initial attack. More than 60% of CNS CD4⁺ cells were of a CD44^high, CD45RB^low memory/effector phenotype both in active EAE, peak EAE and in remission, contrast to lymph nodes where this phenotype never constituted more than 17%. The proportion of CD8⁺ T cells was not increased in remitted mice, and we detected no TCRγδ⁺ cells within the CNS. Our findings demonstrate an overt loss of CD4⁺ T cells from the CNS and the maintenance of an activated state by T cells within the CNS and during remission from EAE. This argues against downregulation of T cell function as a mechanism for remission.     

Paralysis of CD4(+)CD25(+) regulatory T cell response in chronic autoimmune encephalomyelitis

Increasing evidence strongly suggest that CD4(+)CD25(+) regulatory T (Treg) cells play a pivotal role in suppressing the development of autoimmune diseases. However, it remains poorly understood how these cells are involved in the persistence of, or recovery from, the diseases. In the present study, we examined the role of CD4(+)CD25(+) Treg cells in chronic EAE and compared the results with those obtained in acute EAE. In EAE lesions, CD25(+) cells decreased rapidly at the beginning of chronic EAE, whereas these cells were maintained at high levels during the recovery from acute EAE. The number of Foxp3(+)CD4(+)CD25(+) Treg and levels of Foxp3 mRNA in the lymphoid organ were significantly lower in chronic EAE. Importantly, the regulatory function of individual CD4(+)CD25(+) Treg cells was maintained in animals with chronic EAE. Furthermore, adoptive transfer of activated CD4(+)CD25(+) Treg cells suppressed the development of chronic EAE. These findings suggest that impairment of the CD4(+)CD25(+) Treg response is critical for development of chronic autoimmune diseases, and can be adjustable by autologous Treg transplantation.     

Treatment of relapsing experimental autoimmune encephalomyelitis with T cell receptor peptides

Restricted T cell receptor (TCR) VB gene usage by T cells for recognition of antigens involved in the production of experimental autoimmune encephalomyelitis (EAE) offers the possibility of selective immunotherapy. We determined the preferential VB gene usage of lymph node-derived clones from SJL/J mice to recognize the encephalitogenic epitope PLP 139-151 and from PL/J mice to recognize the newly described encephalitogenic epitope PLP 43-64. In addition, the VB gene usage for recognition of PLP 139-151 by T cell lines derived from SJL/J spinal cords was analyzed. Lymph node-derived SJL/J lines and clones specific for PLP 139-151 expressed VB2, VB4, and VB17a preferentially, and PL/J lines and clones specific for PLP 43-64 expressed VB2 and VB8.2 preferentially. A VB4+ SJL/J clone and a VB8.2+ PL/J clone were encephalitogenic. Encephalitogenic SJL/J lines derived from spinal cord expressed VB2, VB10, VB16, and VB17a preferentially, with a predominance of VB2. Candidate TCR peptides were synthesized and tested from the VB gene families VB4, VB8.2, and VB17a, based on our data and previous data on BP-induced EAE in mice. Treatment of relapsing EAE (R-EAE) in SJL/J mice with VB4 and VB17a peptides reduced clinical and histological disease severity, and treatment of R-EAE in (PLxSJL)F1 mice with VB4 and VB8.2 peptides also reduced clinical and histological disease. The use of TCR peptide therapy may have applications for the treatment of human autoimmune diseases such as multiple sclerosis. © 1993 Wiley-Liss, Inc.     

Antigen therapy of experimental autoimmune encephalomyelitis selectively induces apoptosis of pathogenic T cells

Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.     

Suppressor role of rat CD8+CD45RClow T cells in experimental autoimmune uveitis (EAU)

To determine whether decreased regulatory T cell activity contributes to the pathogenesis of recurrent experimental autoimmune uveitis (EAU), we compared the immunoregulatory activity of CD8+CD45RClow T cells isolated from rats that had recovered from acute EAU with those from rats with the progressive, recurrent disease. Our results showed that CD8+CD45RClow T cells isolated from the recovered rats showed suppressive activity in vitro, whereas those from rats with progressive, recurrent EAU do not. Depletion of CD8+CD45RClow T cells from T cells used for adoptive transfer of EAU increased the pathogenic activity of the T cells. Co-transfer of CD8+CD45RClow T cells with uveitogenic T cells prevented the relapse of disease in the recipient rats. The suppressive CD8+CD45RClow T cells expressed increased levels of Foxp3 after stimulation in vitro with the autoantigen, and inhibited the production of IFN-gamma by autoreactive T cells. Our data indicate that the decreased suppressive activity of CD8+CD45RClow T cells is correlated with disease development in this autoimmune disease. Further studies on the biology of this T cell population should provide much needed insights into disease pathogenesis.     

Defective CD4T cell priming and resistance to experimental autoimmune encephalomyelitis in TNF-deficient mice due to innate immune hypo-responsiveness

We report here that tumor necrosis factor (TNF) deficiency causes innate hypo-responsiveness to a broad range of bacterial or viral constituents. In vivo hypo-responsiveness of TNF-deficient mice to mycobacteria results in defective CD4+ T cell priming to antigens administered in complete Freund's adjuvant (CFA). This deficiency is restored by supplementary mycobacteria. Furthermore, we show that even when self-reactive CD4+ T cell priming is fully restored, susceptibility of TNF-deficient mice to experimental autoimmune encephalomyelitis (EAE) depends on the co-administered pertussis toxin (PTx). TNF-deficient mice are completely resistant to EAE at sub-optimal doses of PTx, while supplementary PTx restores susceptibility. Therefore, TNF shows distinct functions in linking innate responsiveness to CD4+ T cell priming and to the induction of autoimmune disease.     

黏附分子CD44在实验性自身免疫性脑脊髓炎中的作用

目的探讨黏附分子CD44在实验性自身免疫性脑脊髓炎(EAE)发病中的作用。方法将20只大鼠随机分为正常对照组及EAE组,EAE组采用粗制髓鞘碱性蛋白(MBP)抗原注入大鼠后足掌皮下(0. 2 ml/100 g)制作EAE模型,观察大鼠的发病情况及病理表现;并采用免疫组织化学法检测两组大鼠脑组织CD44的含量。结果正常对照组大鼠未发病,EAE组大鼠均有不同程度的发病。HE染色后,光镜下观察,正常对照组大鼠脑和脊髓无异常; EAE组大鼠可见脑及脊髓实质内小血管充血,小静脉周围有大量炎性细胞浸润,血管周围白质脱髓鞘改变。免疫组化显示,正常对照组大鼠脑和脊髓组织未发现CD44阳性细胞; EAE组大鼠中枢神经系统(CNS)白质及灰白质交界处可见大量CD44阳性细胞。结论 EAE模型中存在黏附分子CD44的高表达,其对EAE的发病可能起到促进作用。     

黏附分子CD44在实验性自身免疫性脑脊髓炎中的作用

目的 探讨黏附分子CD44在实验性自身免疫性脑脊髓炎（EAE）发病中的作用。方法 将20只大鼠随机分为正常对照组及EAE组,EAE组采用粗制髓鞘碱性蛋白（MBP）抗原注入大鼠后足掌皮下（0.2 ml/100 g）制作EAE模型,观察大鼠的发病情况及病理表现;并采用免疫组织化学法检测两组大鼠脑组织CD44的含量。结果 正常对照组大鼠未发病,EAE组大鼠均有不同程度的发病。HE染色后,光镜下观察,正常对照组大鼠脑和脊髓无异常;EAE组大鼠可见脑及脊髓实质内小血管充血,小静脉周围有大量炎性细胞浸润,血管周围白质脱髓鞘改变。免疫组化显示,正常对照组大鼠脑和脊髓组织未发现CD44阳性细胞;EAE组大鼠中枢神经系统（CNS）白质及灰白质交界处可见大量CD44阳性细胞。结论 EAE模型中存在黏附分子CD44的高表达,其对EAE的发病可能起到促进作用。     

Intra-CNS activation by antigen-specific T lymphocytes in experimental autoimmune encephalomyelitis

Identification and quantitation of autoreactive T lymphocytes is crucial in order to understand the pathogenesis of autoimmune diseases. We used flow cytometry to analyze autoantigen-specific T cellular responses in the well characterized rat experimental autoimmune encephalomyelitis (EAE) model. Cells isolated from both the central nervous system (CNS) tissue and peripheral lymph nodes were analyzed directly ex vivo or after short term in vitro culture with specific autoantigen. CNS infiltrating T lymphocytes displaying an interferon-gamma response to selected encephalitogenic myelin protein epitopes were measured kinetically during an individual disease episode and also between relapses in a chronic rat EAE model. One of the EAE models used displays a restriction towards TCRBV8S2 chain usage by the encephalitogenic T cells. In this model, in vitro production of intracellular interferon-gamma was selectively detected within this T cell subset derived from both the CNS and peripheral lymph nodes. Furthermore, antigen-specific cells infiltrating the CNS in this model produced several-fold higher amounts of interferon-gamma upon antigen stimulation and displayed a significantly increased in vivo proliferation compared with peripheral lymphocytes. These data thus directly demonstrates that T cells stimulated by a specific autoantigen in the periphery primarily acquire effector functions in the cellular environment of the target organ of the autoantigen.     

K+ channel alterations in the progression of experimental autoimmune encephalomyelitis

Voltage-gated K(+) (Kv) channels play critical roles not only in regulating synaptic transmission and intrinsic excitability of neurons, but also in controlling the function and proliferation of other cells in the central nervous system (CNS). The non-specific Kv channel blocker, 4-AminoPyridine (4-AP) (Dalfampridine, Ampyra?), is currently used to treat multiple sclerosis (MS), an inflammatory demyelinating disease. However, little is known how various types of Kv channels are altered in any inflammatory demyelinating diseases. By using established animal models for MS, experimental autoimmune encephalomyelitis (EAE), we report that expression and distribution patterns of Kv channels are altered in the CNS correlating with EAE severity. The juxtaparanodal (JXP) targeting of Kv1.2/Kvβ2 along myelinated axons is disrupted within demyelinated lesions in the white matter of spinal cord in EAE. Moreover, somatodendritic Kv2.1 channels in the motor neurons of lower spinal cord significantly decrease correlating with EAE severity. Interestingly, Kv1.4 expression surrounding lesions is markedly up-regulated in the initial acute phase of both EAE models. Its expression in glial fibrillary acidic protein (GFAP)-positive astrocytes further increases in the remitting phase of remitting-relapsing EAE (rrEAE), but decreases in late chronic EAE (chEAE) and the relapse of rrEAE, suggesting that Kv1.4-positive astrocytes may be neuroprotective. Taken together, our studies reveal myelin-dependent and -independent alterations of Kv channels in the progression of EAE and lay a solid foundation for future study in search of a better treatment for MS.     

Bladder dysfunction in mice with experimental autoimmune encephalomyelitis

The vast majority of patients with multiple sclerosis (MS) develop bladder control problems including urgency to urinate, urinary incontinence, frequency of urination, and retention of urine. Over 60% of MS patients show detrusor-sphincter dyssynergia, an abnormality characterized by obstruction of urinary outflow as a result of discoordinated contraction of the urethral sphincter muscle and the bladder detrusor muscle. In the current study we examined bladder function in female SWXJ mice with different defined levels of neurological impairment following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of central nervous system inflammation widely used in MS research. We found that EAE mice develop profound bladder dysfunction characterized by significantly increased micturition frequencies and significantly decreased urine output per micturition. Moreover, we found that the severity of bladder abnormalities in EAE mice was directly related to the severity of clinical EAE and neurologic disability. Our study is the first to show and characterize micturition abnormalities in EAE mice thereby providing a most useful model system for understanding and treating neurogenic bladder.