Bromodeoxyuridine and light treatment deletes effector but not suppressor cells of autoimmune encephalomyelitis

Spleen cells (SpC) from Lewis rats that have recovered from experimental autoimmune encephalomyelitis (EAE) confer protection against EAE to naive syngeneic recipients if transferred directly (without culture), but transfer EAE if first activated in culture in the presence of myelin basic protein (MBP) antigen. In order to test the hypothesis that both effector (Te) and suppressor (Ts) cells of EAE coexist in recovered rats, but only the Te proliferate in culture in response to MBP, bromodeoxyuridine (BUdR) was added to the culture and dividing cells were killed by exposure to light prior to adoptive transfer. Recipients of BudR + light-treated cells did not develop EAE, showing that Te were deleted by the treatment. In contrast, Ts activity persisted because these recipients were protected against EAE when challenged with an encephalitogenic dose of MBP.     

Clonal diversity of basic protein specific T cells in Lewis rats recovered from experimental autoimmune encephalomyelitis

T cell lines selected from Lewis rats recovered from experimental autoimmune encephalomyelitis (EAE) respond not only to the immunodominant 72-89 epitope of basic protein (BP), but also to secondary epitopes including the I-A restricted 43-67 region of guinea pig (Gp) BP and the I-E restricted 87-99 sequence of rat (Rt) BP. The current study demonstrates at the clonal level the diversity of T cell responses to Gp- and Rt-BP in EAE-recovered rats. As predicted from the response pattern of BP-selected T cell lines, T cell clones from the lines responded to both the dominant and secondary epitopes of BP. In addition, a new majority clonal type was identified that responded to whole BP but not to epitopes represented on enzymatic cleavage fragments or synthetic peptides spanning the BP molecule. Clones representative of each of the three types of Gp-BP responses were characterized for phenotype, major histocompatibility complex restriction, and biologic activity in vivo. All of the clones were strongly CD4+ and co-expressed CD8 at modest levels as measured by both immunofluorescence and Northern blots. All three T cell specificities were I-A restricted. However, only the 72-89 responsive clone could transfer clinical EAE, due most likely to its unique ability to respond to Rt-BP. In contrast, the Gp-BP 43-67 reactive T cell clone transferred protection against EAE, whereas the whole Gp-BP reactive clone transferred delayed-type hypersensitivity response but was neither encephalitogenic nor protective. Thus, the recovery process from EAE is distinguished by an increased diversity of protective clones as well as innocuous clones that may be spawned as encephalitogenic T cells are regulated.     

The proximal peripheral nervous system is a major site of demyelination in experimental autoimmune encephalomyelitis induced in the Lewis rat by a myelin basic protein-specific T cell clone

Experimental autoimmune encephalomyelitis (EAE) was induced in the Lewis rat by the passive transfer of a cytotoxic CD4⁺ T cell clone specific for the 72–89 peptide of guinea-pig myelin basic protein (MBP). Histological studies on rats with neurological signs showed that inflammation was present in the proximal peripheral nervous system (PNS), namely the spinal roots, as well as in the central nervous system (CNS). The main sites of demyelination were the spinal roots in the PNS, and the spinal cord root entry and exit zones in the CNS. The major involvement of the proximal PNS in autoimmune disease directed at MBP is in marked contrast to EAE induced by immunisation with myelin proteolipid protein, where the inflammation and demyelination are restricted to the CNS. These findings may have implications for the human inflammatory demyelinating diseases including multiple sclerosis, in which MBP is a putative target antigen.     

Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis

On the hypothesis that myelin basic protein isolated with surrounding lipids may constitute an autoantigen in demyelinating diseases we studied the antibody response to the lipid-free and lipid-bound form of myelin basic protein during the course of experimental autoimmune encephalomyelitis induced in rats with either form of protein. Immunization with the lipid-bound form of myelin basic protein induced high titres of antibodies directed to the protein, accompanied by no antibodies to cerebroside 30 days after immunization. Antibodies specifically directed to the lipid-bound form of myelin basic protein were revealed after removal of antibodies recognizing the delipidated myelin basic protein. Anti lipid-bound myelin basic protein antibodies could already be detected at day 10 post-immunization, reaching a maximum at day 20 post-immunization. Demonstrations of antibodies entirely specific for the lipid-bound form of myelin basic protein suggests that this molecule may present epitopes not to be found in its already extensively studied primary structure, possibly the result of conformational changes following lipid binding.     

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein VI. Suppression of adoptively transferred disease and differential effects of oral vs. intravenous tolerization

Antigen-driven tolerance is an effective method of suppressing cell-mediated immune response. We have previously shown that oral administration of myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis (EAE) when it is actively induced by MBP emulsified in complete Freund's adjuvant. In order to further study antigen-driven tolerance is this model, we investigated the effect of oral tolerization on adoptively transferred EAE and compared oral tolerance to intravenously (i.v.) administered MBP in both actively induced EAE and adoptively transferred EAE. Although orally tolerized animals were not protected from adoptively transferred EAE, spleen cells from orally tolerized animals suppressed adoptively transferred EAE when co-transferred with encephalitogenic cells or when injected into recipient animals at a different site at the time encephalitogenic cells were transferred. This suppression was mediated by CD8⁺ T cells, correlated with suppression of DTH responses to MBP, and was associated with decreased inflammation in the spinal cord. Unlike oral tolerization, spleen cells from i.v. tolerized animals did not suppress adoptively transferred EAE when co-transfered with encephalotogenic cells although i.v. tolerized animals were protected from adoptively transferred EAE. MBP peptides were then utilized to further characterize differences between i.v. and oral tolerization in the actively induced disease model. Both orally and intravenously administered MBP suppressed actively induced EAE. However, EAE was only suppressed by prior i.v. tolerization with the encephalitogenic MBP peptide 71–90, but not with the non-encephalitogenic peptide 21–40, whereas prior tolerization with 21–40 did suppress actively induced EAE when administered orally. These results suggest a different mechanism of tolerance is initiated by oral vs. intravenous administered antigen. Specifically, oral tolerization suppresses primarily by the generation of active suppression whereas the dominant mechanism of suppression associated with i.v. tolerization appears most consistent with the elicitation of clonal anergy.     

Regulation of experimental allergic encephalomyelitis : Part 4. Further characterization of postrecovery suppressor cells

Suppressor cells that regulate experimental allergic encephalomyelitis (EAE) are present in lymph nodes of Lewis rats that have recovered from the disease, as demonstrated by adoptive transfer of suppression for at least 108 days following challenge of donor rats with myelin basic protein (BP) in complete Freund's adjuvant (CFA). These suppressor lymph node cells (LNC) inhibit the occurrence of active, but not passive EAE in recipients, suggesting that they function early in the inductive phase of the response to BP. The suppressor cells are nylon-adherent, and suppression can be partially abrogated by depletion of B cells with anti-immunoglobulin (ig) and complement (C). Serum from recovered donors has, at best, only a modest suppressive effect when transfused into syngeneic recipients. These findings suggest that the B cells do not mediate suppression on EAE via production of blocking antibody, but may function directly, perhaps by triggering T cells to become suppressor 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.     

Regulation of experimental autoimmune encephalomyelitis: Inhibition of adoptive experimental autoimmune encephalomyelitis by ‘recovery-associated suppressor cells’

We have previously shown the presence of suppressor cells in Lewis rats at the time of spontaneous recovery from experimental autoimmune encephalomyelitis (EAE). These cells, called ‘recovery-associated suppressor cells’ (RASC), are capable of preventing active EAE and inhibiting in vitro the specific proliferative response of encephalitogenic anti-MBP T cell line cells. The present investigations were undertaken in order to lend support to the hypothesis that RASC play an active role in the recovery. We found that RASC can prevent adoptive EAE when admixed with already activated, but not resting, anti-MBP T cells or when injected into the recipients separately from the encephalitogenic cells. They can also arrest the course of an ongoing disease when injected after the beginning of the clinical signs. This study provides the first direct demonstration of the downregulation of an ongoing EAE by suppressor cells.     

Pathogenic MOG-reactive CD8+ T cells require MOG-reactive CD4+ T cells for sustained CNS inflammation during chronic EAE

XIncreasing evidence supports a role for CD8+ T cells in multiple sclerosis. In an attempt to isolate the contribution of CD8+ T cells in a murine model of MS, we immunized mice with a dominant CD8 epitope MOG37–46, a truncated version of MOG35–55. The data presented here show mild disease induced with MOG37–46, characterized by lower clinical scores, a decrease in CNS infiltration and a decrease in microglial activation. CD8+ T cells reactive to MOG37–46 are pro-inflammatory and traffic to the CNS; however, the presence of CD4+ T cells elicits more severe disease and sustained inflammation of the CNS.     

Characterization of measles virus-induced cellular autoimmune reactions against myelin basic protein in Lewis rats

Subacute encephalomyelitis (SAME) in Lewis rats following infection with a neurotropic measles virus (MV) is associated with a cell-mediated autoimmune response (CMAI) to myelin basic protein (MBP). MBP-selected CD4+ T cell lines both from measles-infected animals as well as from rats challenged with guinea pig MBP (Gp-MBP) had a similar pattern of response in the presence of synthetic peptides to Gp-MBP and specifically responded in vitro only to the encephalitogenic and not the non-encephalitogenic or other control peptides. In primary splenic lymphocyte cultures from SAME animals, however, a low but significant T-cell response was obtained against the non-encephalitogenic peptide S67 (residues 69-81) of the Gp-MBP. Moreover, immunization of MV-infected rats with this peptide induced clinical and histological experimental allergic encephalomyelitis (EAE) in 38% of the animals. The results of the study show that the non-encephalitogenic peptide S67 can be rendered encephalitogenic in rats when an additional stimulus is given in the form of MV infection. The data indicate further that MV infection of the central nervous system (CNS) enhances the susceptibility of the CNS to autoimmune T cell aggression.     

牛脊髓髓鞘碱性蛋白的提取及初步鉴定

目的探索一条从牛脊髓中提取牛髓鞘碱性蛋白(M BP)的行之有效的方法,为实验性自身免疫性脑脊髓炎(EAE)动物模型的建立及研究工作奠定基础。方法组织匀浆器将新鲜的牛脊髓捣碎,用London法抽提,考马司亮蓝法鉴定粗提品的含量,SDS-PAGE观测其分子量,M BP免疫W istar大鼠,以鉴定其抗原性。结果粗提品蛋白含量达10m g/m l,粗提品诱导EAE症状典型,发病率高。结论采用London法可从牛脊髓中获取大量的M BP,与其它方法比较产量高,诱导EAE效果好,且本法简便,所以有一定的推广价值。     

Adoptive transfer of experimental allergic encephalomyelitis: Recipient response to myelin basic protein-reactive lymphocytes

We have used adoptive transfer of myelin basic protein (MBP)-reactive lymphocytes in the Lewis rat model of experimental allergic encephalomyelitis (EAE) to identify stages of effector cell development and to investigate the nature of the subsequent recipient response to the transferred cells. Depending on the timing of cell collection, lymph node cells (LNC) obtained from MBP-CFA (MBP emulsified in complete Freund's adjuvant)-immunized donors may directly transfer clinical disease; however, independent of disease development, recipients of LNC develop early onset of clinical disease following immunization of the recipients with MBP-CFA, consistent with the presence of MBP-memory cells in the LNC transfer inoculum. Similarly obtained spleen cells do not directly transfer disease and do not contain MBP-memory cells (as defined by the early onset of clinical disease following MBP-CFA challenge). Spleen cells adoptively transfer clinical disease only following in vitro culture stimulation with antigen or selected mitogens. Recipients of the primary culture-derived encephalitogenic spleen cells also develop an accelerated onset of clinical disease following MBP-CFA challenge, indicative of the presence of MBP-memory cells, and are not vaccinated. Encephalitogenic T cell lines adoptively transfer clinical disease, and in most cases recipients are vaccinated to MBP-CFA-induced active disease, but remain susceptible to adoptively transferred disease. Co-transfer of encephalitogenic T cell line cells with MBP-reactive lymph node or encephalitogenic spleen cells does not alter the vaccination response. We have found that during the process of T cell line development, the vaccinating phenotype is acquired following the second antigen stimulation cycle. These studies also demonstrate that regulation induced by T cell vaccination blocks the development of effector cells from precursor cells and that such regulation is also equally effective in blocking disease development in recipients which have increased numbers of memory cells. Thus, the response to T cell vaccination, once established, is fully capable of inhibiting the development of effector cells from increased numbers of precursor/memory cells, a response that would be needed in the clinical application of vaccination-induced resistance.     

Competitive dissociation of encephalitogenic complexes between antigen presenting cells and myelin basic protein

Splenic T cells from myelin basic protein (MBP)-immunised Lewis rats were activated to transfer experimental autoimmune encephalomyelitis (EAE) by co-culture with MBP-pulsed lymphoid dendritic cells (DC). MBP-pulsed DC could be kept for at least 24 h at 37 degrees C in antigen-free medium without affecting their ability subsequently to activate encephalitogenic T cells. However, MBP-pulsed DC were rendered much less stimulatory after a 6 h, but not 2 h, secondary incubation with ovalbumin. Thus, although encephalitogenic complexes between MBP and DC appear very stable in the absence of competing antigens, in their presence, antigen exchange can take place over a period of a few hours; this has positive implications for therapy of EAE by antigen competition.     

Differential recognition of sequences within the encephalitogenic region of myelin basic protein capable of eliciting cell-mediated immune responses in experimental autoimmune encephalomyelitis

The fine specificity of myelin basic protein (MBP) epitopes capable of eliciting in vivo delayed-type hypersensitivity responses in Lewis rats with experimental autoimmune encephalomyelitis (EAE) was compared to those eliciting in vitro antigen-specific T cell proliferation and augmentation of disease transfer. Utilizing a panel of synthetic peptides with sequences representing the 68–86 region of guinea pig (GP-) or bovine myelin basic protein (B-MBP), animals were primed with one species of peptide and subsequently challenged with either the same peptide or peptides with truncations or substitutions representative of the other species of MBP. In regard to minimal length sequences capable of eliciting delayed-type hypersensitivity (DTH), rats primed with GP-MBP and complete Freund's adjuvant (CFA) exhibited a hierarchical pattern of responsiveness to challenge with a series of truncated peptides, ranking as follows: GP-68–86 > GP-72–86 > GP-68–84 > > GP-75–86 = no activity. This response pattern corresponds to that previously reported for T cell proliferation and activation for disease transfer. Furthermore, a comparison of these T cell-mediated immune parameters, as elicited by the substituted peptides, revealed the response patterns of DTH reactivity to be similar to that previously described for in vitro T cell proliferation with significant DTH responses generated only by the peptide species for which the animal was primed. In contrast, a cross-reactive pattern of recognition was observed in cells mediating disease transfer, with all four 68–86 sequences capable of augmenting activation for adoptive transfer of disease, regardless of the peptide species for which the animal was primed. The differential antigen recognition patterns observed for these EAE-associated immune responses supports the hypothesis that multiple T_H cell subsets are involved in disease pathogenesis.     

T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4⁺ T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, ‘resistant’ strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analyis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonnstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.     

Suppression of clinical weakness in experimental autoimmune encephalomyelitis associated with weight changes, and post-decapitation convulsions after intracisternal-ventricular administration of 6-hydroxydopamine

Selective depletion of central nervous system norepinephrine (NE) by the neurotoxin 6-hydroxydopamine (6-OHDA) in rats subsequently inoculated with myelin basic protein (MBP) and complete Freund's adjuvant (CFA) produced experimental autoimmune encephalomyelitis (EAE) without the usual expected degree of weakness. The preservation of strength occurred in spite of continued weight loss. Post-decapitation myoclonic convulsive kick latency and kick number, which are known to depend on spinal cord NE, agreed well with the degree of weakness through the clinical disease course. The only difference between EAE groups was that the stronger 6-OHDA pretreated EAE animals did not have an elevated pons-medulla NE compared to saline intracisternal-ventricular (i.c.v.) pretreated controls. We conclude that 6-OHDA can influence the clinical course of weakness by interfering with central noradrenergic activity independent of other features associated with disease in EAE. This effect of 6-OHDA may be exerted through alteration of the blood-spinal cord barrier function and/or central nervous system blood flow.     

Treatment of chronic relapsing experimental allergic encephalomyelitis with the intravenous administration of splenocytes coupled to encephalitogenic peptide 91–103 of myelin basic protein

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) is an autoimmune demyelinating disease of the central nervous system and serves as an experimental model of human multiple sclerosis. Amino acid residues p91–103 of myelin basic protein are encephalitogenic in SJL mice and transfer of T cell lines that recognize this epitope results in CR-EAE. We show here that coculture of T cells in the presence of p91–103 that has been chemically cross-linked to the antigen presenting cells renders the T cell lines tolerant to the antigen. Injection of p91–103 coupled splenocytes into animals that had received encephalitogenic p91–103 reactive T cells significantly reduced the incidence and severity of EAE. Furthermore, treatment of mice with a single injection of antigen coupled splenocytes after they had recovered from their initial paralytic attack prevented the development of subsequent clinical relapses in all animals. These studies indicate than this effect is long lasting and can be successfully accomplished in an established autoimmune disease. Hence this form of immunotherapy may be considered as a therapeutic modality in the treatment of autoimmune diseases when the autoantigens are known.     

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。     

Pathogenic and non-pathogenic T lymphocytes specific for the encephalitogenic epitope of myelin basic protein: Functional characteristics and vaccination properties

Activated CD4⁺ T lymphocytes specific for myelin basic protein (MBP) can cause experimental autoimmune encephalomyelitis (EAE) upon their inoculation into syngeneic recipients. In Lewis rats, most of the pathogenic T cell clones that develop following immunization with MBP are reactive against the 72–84 amino acid sequence of MBP, the major encephalitogenic region for Lewis rats. In this study, some MBP-specific T cell clones were found to be non-pathogenic, in spite of their strong reactivity against the encephalitogenic epitope. One of these non-pathogenic clones, designated Znp, and an encephalitogenic clone, Z1a-p, were derived from Z1a encephalitogenic line cells. These subclones were compared for epitope specificity, T cell receptor variable gene expression and for various functional activities, in order to delineate properties crucialof pathogenicity. The Z1a-p and Znp cells expressed comparable levels of the T cell receptor genes and shared strong reactivity against the 72–84 epitope of MBP. The pathogenic Z1a-p cells displayed MBP-specific cytolytic activity in vitro, provided an in-vivo ‘help’ for elicitation of MBP-specific antibodies, mediated a delayed type hypersensitivity (DTH) response to MBP, caused EAE and vaccinated against the disease, thus demonstrating that a single CD4⁺ T cell clone is capable of eliciting various functions. The non-pathogenic Znp cells could also carry out most of these various functions, but failed to mediate a DTH response to MBP in normal animals. However, when inoculated into sublethally (650 R) irradiated syngeneic recipients, the Znp cells became highly pathogenic and mediated DTH response to MBP. Local irradiation of the recipient facilitated a DTH response to MBP in the irradiated ear, indicating that Znp cells are equipped with the effector mechanisms required for pathogenicity, and that their failure to cause disease may be accounted for by their inability to migrate into extravascular target tissue. Similar data were obtained with an independently isolated non-pathogenic clone, LB-3, specific for the encephalitogenic epitope of MBP. The ability of these non-pathogenic cells to vaccinate against EAE mediated by pathogenic cells raises the possibility that such non-pathogenic cells may play a role in triggering downregulation of pathogenic T cells.     

T cell immunity to myelin basic protein induces anterior uveitis in Lewis rats

Uveitis of unknown etiology is known to occur in association with various systemic disorders. We now report that anterior uveitis (AU) can be produced by T cell immunity to myelin basic protein (BP) and accompanies experimental autoimmune encephalomyelitis (EAE). EAE with AU was induced in Lewis rats by immunization to BP in CFA or by immunization to various BP peptides including the encephalitogenic 71–90 peptide. Slit-lamp biomicroscopy of BP-immunized Lewis rats revealed AU, characterised by inflammation of the iris, in 73% of the eyes. The onset of AU in actively immunized rats varied between days 12 and 26, often appearing after spontaneous remission of the paralysis, the hallmark of EAE. The course of AU was progressive, affecting more than 50% of the surface of the iris in 16 of 29 diseased eyes. Like the paralysis, the AU was self-limiting: within 2 weeks the disease remitted. In addition, AU could be adoptively transferred to naive and irradiated rats by a T cell clone specific for BP peptide 71–90. The present observations are compatible with the idea that AU may be triggered by BP-reactive T cells. The myelinated nerves present in the iris have been shown to contain BP. However, these peripheral nerves would now appear to be the only peripheral nerves susceptible to acute EAE.