Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis

Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9/30 for MAG, 0/17 for PLP, and 8/81 for MBP. No control subjects responded to MAG or PLP, and only 1/29 control subjects responded to MBP. Three of the patients that responded to MAG also responded to MBP. Although the mean proliferative response to MAG and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The OND group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to MAG (P less than 0.05), and OND vs. controls and active MS that responded to PLP (P less than 0.025). The greatest individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases.     

T cell responses to myelin basic protein in patients with spinal cord injury and multiple sclerosis.

Autoimmune inflammation secondary to myelin destruction may play an inhibitory role in restoration of nerve functions in spinal cord injury (SCI). In this study, we demonstrated that T cells recognizing myelin basic protein (MBP) occurred at a high precursor frequency in patients with SCI, which was compatible to that in patients with multiple sclerosis (MS), a disease of presumed autoimmune pathology. The findings suggest of hyperactivity of MBP-reactive T cells in patients with SCI. MBP-reactive T cell lines derived from patients with SCI exhibited a preferential recognition pattern toward the 81-99 and the 151-169 regions of MBP. There were functional differences in the epitope recognition and cytokine profile between two panels of MBP-reactive T cell lines derived from patients with SCI and patients with MS. The study provides new evidence important for further investigation of the role of the inflammatory component in SCI.     

亚低温治疗对实验性脑出血大鼠死亡率及脑组织钙含量的影响

目的本研究观察３２℃亚低温对实验性脑出血大鼠２４ｈ内死亡率和脑组织钙含量的影响。方法１３４只大鼠分成两部分：（１）６８只大鼠用于死亡率观察;（２）６６只大鼠用于脑组织钙含量测定。每一部分分成假手术对照组、常温脑出血组及亚低温脑出血组。结果常温组２４ｈ内死亡率为３６．６７％,亚低温组为４．５５％;脑组织钙含量常温组较对照组和亚低温组为高。结论亚低温治疗能显著减少实验性脑出血大鼠２４ｈ内死亡率,减少脑出血后脑组织钙含量。     

Sensitization of cerebrospinal fluid and peripheral blood lymphocytes to myelin basic protein in multiple sclerosis

Cerebrospinal fluid (CSF) and peripheral blood (PB) lymphocyte sensitization to rabbit myelin basic protein (MBP) in 44 multiple sclerosis (MS) patients, 21 patients with other neurological diseases (OND) and 14 persons with neurosis was studied with the antigen-active rosette forming cells (Ag-ARFC) assay. The frequency of sensitization of CSF lymphocytes to MBP in groups of MS patients in the relapse stage and the chronic progressive stage was higher than in the group of MS patients in the stable stage and the OND patients. None of the healthy subjects showed a positive reaction with MBP. In BP there were no differences in the incidence of sensitization to MBP between patients in various stages of the disease, but it was higher than in the group of patients with OND and neuroses. In the patients who had suffered from MS for less than 4 years, sensitization to MBP was more common in CSF lymphocytes than in BP lymphocytes. The results suggest that primary sensitization to MBP occurs in CSF, and is probably secondary to myelin damage. However at present it is difficult to determine the extent to which sensitization of CSF and PB lymphocytes to MBP play a role in further demyelination processes.     

Response of human T lymphocyte lines to myelin basic protein: association of dominant epitopes with HLA class II restriction molecules

In animals, the selection in vitro of T cell lines to myelin basic protein (MBP) can define immunodominant and encephalitogenic epitopes which are preferentially associated with class II major histocompatibility (MHC) molecules. These principles were used to evaluate the specificity and MHC restriction of 14 human MBP-reactive T cell lines selected from normal individuals and patients with multiple sclerosis (MS) and other neurological diseases (OND). The four normal T cell lines recognized single, separate immunodominant MBP epitopes which were restricted by MHC molecules from the DR or in one case the DP class II locus. In contrast, the MS and OND T cell lines recognized multiple MBP epitopes, each in association with a discrete class II MHC molecule from the DR or DQ locus. Overall, HLA-DR molecules were used preferentially to associate with epitopes on human MBP, restricting 26/33 responses. As predicted from animal studies, T cells from genetically disparate individuals responded to different immunodominant epitopes on human MBP in association with distinct MHC class II molecules. HLA-DR2, which is overrepresented in MS patients, possessed an unusual capacity to restrict all eight epitopes identified on MBP in this study. These data provide the first evidence of genetically restricted human T cell recognition of potentially encephalitogenic epitopes of MBP.     

The immune response to a subdominant epitope in myelin basic protein exon-2 results in immunity to intra-and intermolecular dominant epitopes

Experimental autoimmune encephalomyelitis was induced in SJL/J mice by adoptive transfer of a MBP exon-2 peptide-specific T cell line. The T cell line, when tested for antigen specificity, reacted strongly with exon-2 peptide, but not with MBP peptides pAc1-11, p43–88, p89–101 or PLP p139–151. The specificity of splenic or lymph node T cells isolated from mice with acute or first relapse EAE induced by adoptive transfer of the exon-2-specific T cell line was identical to the transferred line. Splenocytes or lymphocytes isolated from mice at the second relapse were reactive with MBP p43–88, p89–101 and PLP p139–151 in addition to exon-2 peptide and MBP peptide Ac1-11. T cell lines selected by culture with MBP exon-2 peptide or PLP p139–151 from splenic cells from mice with relapsing EAE were weakly encephalitogenic; however, T cell lines selected from the same mice with MBP pAc1-11 were not encephalitogenic. T cells from the exon-2 and p139–151 T cell lines primed recipients for rapid onset severe EAE, whereas the pAc1-11 T cell line did not. T cells from the exon-2-specific line did not express V_β17a⁺ TCR; however, peptide-specific T cell lines derived from the spleens of relapsing animals did express this TCR gene segment providing direct evidence of recruitment and sensitization of recipient T cells.     

Binding properties of cerebrospinal fluid IgG in multiple sclerosis and other neurological diseases

A solid phase radioimmunoassay (RIA) was used to detect antibodies to myelin or myelin basic protein (MBP) in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or other neurological diseases (OND). When measured at the same IgG concentration, MS samples had higher binding values than OND against myelin, but not against MBP. Using F(ab')2 fragments purified from pools of MS and OND CSF there was no difference in binding to myelin between MS and OND samples. These results indicate that anti-MBP antibodies are nt a feature of MS and binding of CSF IgG to myelin is not due to specific antibody, but is probably the result of non-specific binding to Fc receptors.     

IgG reactivity against citrullinated myelin basic protein in multiple sclerosis.

An increased level of citrullinated myelin basic protein (MBP-C8) has been reported in the brains of multiple sclerosis (MS) patients. However, the involvement of the immune response to post-translational modified MBP in the pathophysiology of MS remains speculative. The aim of this study was to compare the levels of immunoglobulin G antibodies to several MBP epitopes, before and after citrullination, in the cerebrospinal fluid (CSF) and sera of MS patients using enzyme-linked immunosorbent assay (ELISA). We analyzed antibody reactivity against various MBP-peptides in the CSF and sera of 60 MS patients, and 30 patients with other neurological diseases (OND) as controls. The peptides tested were: MBP(75-98) (peptide 1), native (peptide 2) and citrullinated (peptide 3) MBP(108-126) (ARG(122)-->Cit(122)), and native (peptide 4) and citrullinated (peptide 5) MBP(151-170) (ARG(159, 170)-->Cit(159, 170)). All selected peptides could support an immune reactivity in CSF and sera of MS and OND patients. A higher reactivity against peptide 4 was found in the CSF of MS patients compared with OND patients (P<0.0001), but not against citrullinated peptides (peptides 3 and 5). However, we observed that the citrullination state of peptide 2 modified the patterns of immune reactivity more markedly in MS patients (P<0.0001) than in OND patients (P<0.02). Although some MBP epitopes could be a potential target in MS, our data did not demonstrate any difference of antibody response to MBP peptides in their citrullinated forms.     

CSF antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases

Cerebrospinal fluid (CSF) from 18 multiple sclerosis (MS) patients, 13 subacute sclerosing panencephalitis (SSPE) patients, 22 other neurological disease (OND) patients, and 7 neurotic patients as controls were tested in an 125I-labeled anti-human F(ab')2 binding assay for the presence of antibodies to normal human brain cells from tissue culture, human fibroblasts, plasma membranes of MS and normal human brain, myelin basic protein (MBP) and bovine oligodendrocytes. Antibodies to MBP and to oligodendrocytes were found in the CSF of MS, SSPE and OND patients. Absorption of CSF with bovine CNS myelin significantly diminished binding activity to oligodendrocytes. Antibodies in the CSF against MBP and oligodendrocytes, on which some myelin determinants are expressed, seem to be a common feature of diseases in which demyelination is a component.     

CSF myelin basic protein in multiple sclerosis

Cerebrospinal fluid (CSF) from 221 patients with multiple sclerosis (MS) and 85 patients with other neurological disorders (OND) was examined using a competitive radioimmunoassay for myelin basic protein (MBP) immunoreactivity. MBP was found in 46 of 55 MS patients (84%) examined within six weeks of relapse but in only 11 of 85 patients (13%) with OND. There was a significant correlation between the concentration of MBP in the CSF and relapse severity in patients seen within four weeks of the onset of symptoms (p less than 0.01). Of 44 patients in remission, MBP was detected in 12, and these patients had a significantly higher tendency to subsequent relapse (p less than 0.05). In 72 patients with progressive disease the presence of MBP in the CSF reflected the confidence of clinical diagnosis. The results of this study suggest that measurement of MBP in the CSF gives an objective method of monitoring disease activity in patient with MS.     

Sequence 104–117 of myelin proteolipid protein is a cryptic encephalitogenic T cell determinant for SJL/J mice

Immunization of animals with myelin proteolipid protein (PLP) causes experimental autoimmune encephalomyelitis (EAE), a disease model that shares many features with human multiple sclerosis (MS). The SJL/J (H-2⁵) mouse is widely used in EAE studies because of its high disease susceptibility. Previous studies have shown that sequences 139–151 HCLGKWLGHPDKF and 178–191 NTWTTCQSIAFPSK represent distinct co-immunodominant encephalitogenic determinants of PLP for SJL/J mice. In the present study, we identify a third distinct PLP encephalitogenic peptide for SJL/J mice. Following immunization with PLP 104–117 KTTICGKGLSATVT, 10/14 SJL/J mice developed clinical and histological EAE with a mean time of onset of 38 days (18–65 days). T cell lines generated from SJL/J mice immunized with p104–117 were predominantly (> 90%) CD3⁺, CD4⁺, αβTCR⁺, CD8^dim, γδTCR^dim T cells and responded in an Ag-specific, I-As-restricted manner to p104–117. Upon adoptive transfer of 16−40 × 10⁶ T line cells, EAE was produced in naive SJL/J recipients 20–34 days after transfer. The delayed onset of both active and passive disease may be related to the non-immunodominant, cryptic nature of p104–117 in SJL/J mice. Lymph node cells from SJL/J mice immunized with either whole PLP or with pooled encephalitogenic PLP peptides responded to challenge with the immunodominant PLP determinants p139–151 and p178–191 but did not respond to p104–117. The existence of three distinct PLP encephalitogenic T cell determinants for SJL/J mice suggests that susceptibility to EAE and perhaps MS may be related to promiscuous T cell recognition of multiple myelin protein determinants.     

Optic neuritis and multiple sclerosis: the T cell repertoires to myelin proteins and MBP peptides change with time

Autoreactive T cells recognizing myelin basic protein (MBP), proteolipid protein (PLP) and MBP peptides have been described in multiple sclerosis (MS) and optic neuritis (ON), but their role in disease pathogenesis, if any, is unknown. A consistency of the T cell repertoire over the course of MS and ON should facilitate the development of specific immunotherapies. We have examined the T cell responses to autoantigens in two consecutive blood specimens taken from patients with ON and MS, and in two consecutive CSF specimens obtained from ON patients. As read-out numbers of T cells responding to antigen stimulation by the secretion of interferon-γ were estimated. Pronounced differences in occurrence and numbers of T cells recognizing MBP, MBP peptides with the amino acid sequences 63–88, 110–128 and 148–165, and PLP were noticed in individual ON and MS patients over the course of disease. The MBP peptide among those three included, that was predominantly recognized by T cells in the individual patient, also varied over the course. The quantitative and qualitative changes of the myelin antigen-specific T cell response in MS and in ON, the latter to a certain extent reflecting the situation in early MS, do not favor the future useful development of specific immunotherapies in these diseases.     

Lyme neuroborreliosis: cerebrospinal fluid contains myelin protein-reactive cells secreting interferon-γ

The mechanisms causing neurological manifestations and influencing the outcome in patients infected with the spirochete Borrelia burgdorferi are unknown. To study the involvement of autoimmune mechanisms in patients with Lyme neuroborreliosis (LN), ELIspot assays were used to determine the numbers of T lymphocytes which, upon stimulation with the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) and two MBP peptides, responded by the secretion of interferon-γ (IFN-γ). LN patients had compared to patients with other neurological diseases and tension headache in CSF elevated numbers of MBP- and PLP-reactive T helper type 1 (Th1) like IFN-γ secreting cells at mean frequencies of about 1 per 1300 and 1 per 1500 CSF cells, respectively. Numbers were elevated up to 5 months after successful treatment of LN, and they did not correlate with clinical findings. Autoreactive T cells were not elevated in the LN patients' blood. These findings match the previously reported elevation of anti-MBP IgG antibody secreting cells exclusively in CSF in LN. The IFN-γ release by autoreactive cells and the secretion of autoantibodies in the CSF may have relevance for development and outcome of LN.     

海洛因海绵状白质脑病的脑脊液和血清髓鞘碱性蛋白及其抗体检测及意义

目的　通过对海洛因海绵状白质脑病 (HeroinSpongiformLeukoencephalopathy ,HSLE)患者血清和脑脊液髓鞘碱性蛋白 (myelinbasicprotein ,MBP)及其抗体 (Anti MBP)检测 ,探讨MBP与HSLE的关系。方法　采用酶联免疫吸附法 (ELISA)检测 1 8例HSLE患者、2 3例Heroin成瘾者 (吸毒但无HSLE临床症状者 ) ,1 7例多发性硬化患者(MS组 )、对照组 2 0例 (NC组 )的血清以及脑脊液 (CSF)中MBP及Anti MBP水平。结果　HSLE组、MS组CSF和血清MBP均值明显高于NC组和Heroin成瘾组 (P <0 .0 5) ,HSLE组与MS组CSF、血清MBP均值无统计学差异 (P >0 .0 5) ,Heroin成瘾组血清和CSFMBP均值与NC组间无统计学差异 (P >0 .0 5) ,4组血清和CSF的Anti MBP含量差异不显著 (P >0 .0 5)。结论　HSLE患者CSF及血清中MBP含量增高 ,MBP上升水平与髓鞘损伤程度有关 ,该病的髓鞘存在病理性损害 ,以及血脑屏障 (blood brainbarrier,BBB)的通透性改变。MBP检测可作为HSLE诊断及与海洛因成瘾者鉴别诊断的重要参数 ,它在HSLE病理过程中的作用机制有待进一步研究     

Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein.

Proteolipid protein (PLP) is a major component of the central nervous system (CNS) myelin membrane and has been shown to induce acute experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals. Here we describe conditions by which a relapsing-remitting form of EAE can be reliably induced in SJL/J mice either actively immunized with the major encephalitogenic PLP peptide, PLP13-151(S), or following adoptive transfer of PLP139-151(S)-specific T cells. The disease follows a reliable relapsing-remitting course with acute clinical signs first appearing 6-20 days after priming or transfer and relapses first appearing at 30-45 days. The initial onset of disease correlates with delayed-type hypersensitivity (DTH) reactivity specific for PLP139-151(S), in the apparent absence of T cell reactivity to the major myelin basic protein (MBP) peptide. Histologically, both the active and adoptive forms of the disease are characterized by extensive mononuclear cell infiltration and severe demyelination of the CNS. These results suggest that T cell responses specific for PLP139-151(S) are sufficient to induce clinical and histological R-EAE in SJL/J mice. This model should prove useful for examination of the cellular and molecular events involved in clinical relapses and perhaps in determining the role of PLP-specific T cell responses in multiple sclerosis (MS).     

髓鞘碱蛋白对脑梗死和多发性硬化的诊断价值

目的探讨髓鞘碱蛋白（ＭＢＰ）对脑梗死（ＣＩ）和多发性硬化（ＭＳ）的诊断价值。方法对１１４例ＣＩ患者、２８例ＭＳ患者、２４名正常者血清以及部分患者脑脊液（ＣＳＦ）的ＭＢＰ进行了检测。结果ＣＩ、ＭＳ患者血清和ＣＳＦ的ＭＢＰ均显著高于正常对照组（Ｐ〈０．０１）,ＭＳ组又高于ＣＩ组（Ｐ〈０．０１）;ＭＳ组中病情活动期患者ＭＢＰ又高于缓解期。结论血清和ＣＳＦ的ＭＢＰ检测对ＭＳ和ＣＩ的鉴别诊断,以及判断ＭＳ病     

Autoimmune T cell repertoire in optic neuritis and multiple sclerosis: T cells recognising multiple myelin proteins are accumulated in cerebrospinal fluid.

Monosymptomatic unilateral optic neuritis is a common first manifestation of multiple sclerosis. Abnormal T cell responses to myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG) have been implicated in the pathogenesis of multiple sclerosis. Antigen-reactive T helper type 1 (Th1)-like cells that responded by interferon gamma (IFN-gamma) secretion on antigen stimulation in vitro were counted. Untreated patients with optic neuritis and multiple sclerosis had similarly raised levels of T cells recognising MBP, PLP, and MAG in peripheral blood. Such T cells were strongly enriched in CSF. None of these myelin antigens functioned as immunodominant T cell antigen characteristic for optic neuritis or multiple sclerosis. The autoimmune T cell repertoire was not more restricted in optic neuritis (as an example of early multiple sclerosis). The autoreactive T cell repertoires differed in blood compared with CSF in individual patients with optic neuritis and multiple sclerosis. No relations were found between specificity or quantity of autoreactive T cells in blood or CSF, and clinical variables of optic neuritis or multiple sclerosis, or occurrence of oligoclonal IgG bands in CSF. The role of raised MBP, PLP, and MAG reactive Th1-like cells found in optic neuritis and multiple sclerosis remains unexplained.     

Novel monoclonal antibodies against proteolipid protein peptide 139-151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions

Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with epitopes of the proteolipid protein (PLP), a major myelin constituent, forms a useful model for the study of multiple sclerosis (MS). In addition, MS patients display PLP-specific T- and B-cell responses, suggesting that PLP reactivity is relevant to pathogenesis.Here, the generation and characterization of a panel of mouse monoclonal antibodies (Mab) against PLP139-151, the prominent encephalitogenic sequence in SJL/J mice is described. Five Mab were generated by conventional immunization of an SJL/J mouse and hybridoma generation. These Mab reacted well with the PLP139-151 peptide in ELISA and belonged to the IgG2a and IgG2b subclasses, consistent with CD4+ T helper 1-cell-supported antibody formation. The Mab also efficiently detected PLP peptide-BSA conjugates in Western blot, confirming their multi-assay applicability.The Mab were subsequently used to determine the occurrence of demyelination in brains of MS patients and marmoset monkeys with EAE. Immunohistochemistry on both paraffin and frozen sections demonstrated a homogeneous expression of PLP139-151 in normal myelin, and a complete absence in lesions containing demyelinated areas, confirming that the Mab can be used as a general myelin marker. In active demyelinating MS lesions, the Mab visualized the peptide in the cytoplasm of macrophages containing phagocytosed myelin.In conclusion, this panel of Mab against the encephalitogenic PLP139-151 epitope forms a useful tool for further study of autoantigen expression, demyelination/remyelination and the staging of lesional activity in MS patients, as well as in EAE models in distinct animal species.     

Cytotoxic effects of myelin basic protein-reactive T cell hybridoma cells on oligodendrocytes

Cells of a rat/mouse T cell hybridoma reactive to the encephalitogenic peptide of myelin basic protein (MBP) was found to be cytotoxic to 51Cr-labelled rat oligodendrocytes (oligos) inducing 52 +/- 5% 51Cr release vs. 28 +/- 2% spontaneous 51Cr release from replicate oligos. The hybridoma cells were not toxic for rat astrocytes or concanavalin A-stimulated lymphoblasts. Hybridoma T cells reactive to an experimental allergic encephalomyelitis-irrelevant antigen (ovalbumin) were not cytotoxic to oligos. The cytotoxic reaction required cell-cell contact but did not require the in vitro presence of antigen-presenting cells MBP. The target antigen on the oligos is not yet defined. These studies suggest that MBP-reactive T cells can be directly cytotoxic to oligos in the absence of other cell populations.     

Interferon beta induces T-helper 2 immune deviation in MS

OBJECTIVE: To define the in vitro effects of interferon beta la (IFN-beta1a) on myelin basic protein (MBP)-reactive T cells and to determine its regulatory mechanism on cytokine networks in patients with MS. METHODS: The proliferation and cytokine production of MBP-reactive T-cell clones were measured in thymidine uptake assays and ELISA respectively. The precursor frequency of MBP-reactive T cells was estimated in a microwell culture system. RESULTS: IFN-beta inhibited the proliferation of established MBP-reactive T-cell clones, which correlated with enhanced production of anti-inflammatory interleukin (IL)-4 and IL-10, and a decrease in tumor necrosis factor alpha (TNF-alpha) and IFN-gamma. When examined with peripheral blood mononuclear cells (PBMCs), IFN-beta was found to reduce the in vitro T-cell responses to MBP, as indicated by the significantly decreased frequency of MBP-reactive T cells. The decreased frequency of MBP-reactive T cells corresponded to an augmented production of IL-4 and IL-10. Although the level of TNF-alpha and IFN-gamma was generally unaltered or decreased, IFN-beta appeared to enhance the production of IFN-gamma in PBMCs derived from some individuals with MS. CONCLUSION: Interferon beta la (IFN-beta) suppresses myelin basic protein (MBP)-reactive T cells and induces immune deviation toward the production of T-helper 2 cytokines, which may contribute to its therapeutic benefit in MS. The study also suggests some heterogeneity in MBP-reactive T-cell responses to IFN-beta in different individuals with MS.