Cyclosporin A treatment of an induced attack in a chronic relapsing model of experimental allergic encephalomyelitis

Chronic relapsing experimental allergic encephalomyelitis (EAE) was induced in 8-week-old SJL/J mice by injecting an encephalitogenic emulsion on Day 0 and Day 7. A third injection was given on Day 70 postinoculation (PI) which precipitated an attack with high mortality (62%) after 7-9 days. Cyclosporin A (CsA) was given at doses of 5, 2, and 0.5 mg per mouse, one or three times per week starting from Day 40 PI and continuing over the next 17 days. High serum levels of CsA were measured by radioimmunoassay. However, gross and microscopic pathological examination showed no indication of hepatic or renal toxicity at these doses. In the CsA-treated mice, there was a dose-dependent shortening of the length and severity of the attack forced by challenge with the third injection. The mortality was significantly (P less than 0.05) reduced when compared with the non-CsA-treated controls. In addition, the data demonstrate a decrease of lymphocyte-derived chemotactic factor produced from phytohemagglutinin-stimulated spleen cells of mice with chronic relapsing EAE treated with CsA when compared to normal mice and mice with chronic relapsing EAE treated with vehicle alone. We conclude that it is possible to effect an induced acute attack in ongoing chronic relapsing EAE with CsA treatment.     

Therapeutic activity of leflunomide in acute and chronic relapsing experimental allergic encephalomyelitis

We examined the therapeutic effect of leflunomide in the two models of acute and chronic relapsing EAE in Lewis rats. In the first model, sensitization of adult rats with guinea pig spinal cord resulted in an acute clinical episode of severe EAE, and by day 15 all animals died. Treatment of these sensitized Lewis rats with leflunomide was most effective in delaying and reducing the onset of clinical symptoms and mortality was prevented. The protection afforded by leflunomide was long-lasting and no subsequent relapse has been observed. In the second model of chronic relapsing EAE, aged Lewis rats (6–8 months old) were immunized with rabbit myelin basic protein, and all untreated animals developed a disease with up to three relapses. The second and third episodes were both milder and shorter in duration than the first. All animals treated with leflunomide survived the first attack, which was also delayed, in comparison to untreated controls, and relapses did not occur. Inhibition of pathological signs and prevention of relapses were observed even when leflunomide treatment was started after the first appearance of clinical symptoms of chronic relapsing EAE.     

The origin and specificity of intrathecal IgG in chronic relapsing experimental allergic encephalomyelitis

The source of IgG in the cerebrospinal fluid (CSF) in guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE) was investigated using quotient analysis of total IgG and albumin concentrations and by computing CSF-plasma ratios of specific IgG concentrations. Increased blood-CSF barrier (B-CSFB) permeability was shown by elevated albumin quotients in both relapse and remission phases of CR-EAE and intrathecal production of IgG was indicated by raised ratios of IgG to albumin in the CSF. Intrathecal IgG synthesis was greatest in guinea pigs which had little B-CSFB damage. When enzyme-linked immunosorbent assays (ELISA) for whole cord, myelin basic protein (MBP) or Mycobacterium tuberculosis were performed with CSF and plasma adjusted to the concentration of total IgG, the CSF/plasma ratios of ELISA results for specific antibodies were less then unity and ratios for whole cord and MBP were lower than those for M. tuberculosis. There was thus no evidence for a selective increase in the CSF of antibody specific either for the neuroantigens tested or for adjuvant components. The CSF-plasma ratios for each specific antibody were inversely correlated with the extent of total IgG intrathecal synthesis, suggesting that much of the antibody production within the CNS is the result of polyclonal B cell activation.     

Chemokine upregulation follows cytokine expression in chronic relapsing experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalomyelitis (ChREAE) is an autoimmune disease of the central nervous system (CNS) induced by CNS myelin components. In the early active stage, both ChREAE and multiple sclerosis (MS) are characterized by the presence of perivascular inflammatory cuffs disseminated in the CNS. There is growing evidence that chemoattractant cytokines (chemokines) play an important role in this process. The main goal of the present study was to analyse the hypothesis that chemokine expression in the CNS during autoimmune inflammation is regulated by proinflammatory cytokines. To address this concept, we analysed temporal relations between chemokine and cytokine expression during ChREAE. Phasic upregulation of gene expression for chemokines T-cell activation gene 3 (TCA-3)/CCL1, monocyte chemoattractant protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-1 alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4, regulated on activation normal T cell expressed and secreted (RANTES)/CCL5 and MIP-2/CXCL2-3 as well as cytokines tumour necrosis factor-alpha (TNF-alpha), -beta, LT-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) in the CNS was observed during attacks of ChREAE. Expression of cytokines TNF-beta and LT-beta preceded, and the expression of TGF-beta1 followed chemokine upregulation. Our results suggest that chemokine expression during CNS autoimmune inflammation may be regulated by some proinflammatory cytokines.     

Cerebrospinal fluid interleukin 1 like activity during chronic relapsing experimental allergic encephalomyelitis.

Cerebrospinal fluid (CSF) and plasma samples were taken from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE). The samples were assayed for interleukin 1 (IL-1) in the C3H/Hej mouse thymocyte assay. After removal of inhibitors, IL-1 was detectable in low amounts in plasma (5 U/ml) throughout the course of the disease but was raised in the acute phase (12 U/ml). CSF IL-1 was, however, only present in low amounts (6 U/ml) during the acute phase but was elevated (18 U/ml) during the chronic stages of CREAE. During the relapse phase levels of IL-1 correlated with the total leucocyte count in the CSF. On gel filtration of CSF, IL-1 activity eluted at approximately 15 kD and could not be attributed to leakage of plasma IL-1 during CSF puncture or IL-2 activity.     

Inhibition of chronic relapsing experimental allergic encephalomyelitis in the mouse by the alkyl-lysophospholipid ET-18-OCH3

The effect of the anti-tumour agent alkyl-lysophospholipid (ALP) ET-18-OCH3 on the development of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the mouse was investigated. Experimental allergic encephalomyelitis developed in the majority (greater than 96%) of mice immunized with autologous spinal cord homogenate in Freund's complete adjuvant. Alkyl-lysophospholipid, in doses of 25 mg/kg/day or 50 mg/kg/day, inhibited the onset of clinical signs of acute phase CREAE when orally administered starting on the day of disease induction. Similarly if treatment with 50 mg/kg/day was delayed until day 9 post-inoculation the incidence of disease and severity of clinical signs were also significantly reduced (P less than 0.02) as compared with vehicle fed animals. However, when treatment began on day 12, just prior to the onset of clinical disease, although the incidence of disease was not significantly altered the severity of disease was significantly (P less than 0.002) reduced compared with vehicle treated animals. These data suggest that although the major effect of ALP is on the inhibition of the generation of the autoimmune response there appeared to be some therapeutic benefit at a later stage of acute disease. Therefore, this study was extended to the treatment of post-acute phase remission animals. It was found that the oral administration of 50 mg/kg/day marginally reduced and that 75 mg/kg/day significantly (P less than 0.05) reduced the incidence of relapsing disease compared with vehicle treated controls. This suggests that ET-18-OCH3 may have some potential in the treatment of ongoing autoimmune disease of the central nervous system.     

IgG subclass responses and immediate skin sensitivity in guinea-pigs with chronic relapsing experimental allergic encephalomyelitis

In strain-13 guinea-pigs inoculated for chronic relapsing experimental allergic encephalomyelitis (CR-EAE), IgG1 and IgG2 subclass antibody responses were investigated using single radial immunodiffusion and enzyme-linked immunosorbent assays (ELISA) for IgG1 and IgG2 specific for whole cord, myelin, myelin basic protein and Mycobacterium tuberculosis. The early acute stage revealed no increase in IgG1 but was associated with increased levels of IgG2 specific for neural and adjuvant components. Throughout the chronic phase of the disease, there were increased levels of IgG of both subclasses specific for the antigens tested but a preferential synthesis of IgG1. Levels of both IgG1 and IgG2 specific for neuroantigens were lowest in those guinea-pigs which did not develop signs of chronic disease. Immediate skin sensitivity against a wide range of neural antigens was not demonstrated though positive results may have been masked by the ability of myelin basic protein to induce non-specific mast cell degranulation and by altered histamine responsiveness in disease. Guinea-pigs with chronic paralysis had a lower skin sensitivity to histamine, compound 48/80 and M. tuberculosis than those in remission.     

Macrophages in T cell line-mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in Lewis rats.

About 50% of the mononuclear cells in the perivascular lesions in the central nervous system (CNS) of rats suffering from experimental allergic encephalomyelitis (EAE) are blood-borne macrophages. In this study we investigated the role of these macrophages in different variants of EAE, using a liposome-mediated macrophage depletion technique. Intravenously injected liposomes containing dichloromethylene diphosphonate (Cl2MDP) are ingested by macrophages and cause temporary and selective elimination of these cells. Macrophage depletion during EAE induced by a T cell line specific for myelin basic protein (MBP; T cell-EAE) suppresses development of neurological signs of EAE. T cell-EAE with pronounced demyelination as induced by an additionally injected MoAb directed against myelin oligodendrocyte glycoprotein (MOG) was also significantly ameliorated after macrophage depletion. During chronic relapsing EAE (CR-EAE) the occurrence of relapses was prevented or suppressed, provided that the liposomes were injected before the initiation of a putative relapse. A chronic progressive course of CR-EAE was not modified by Cl2MDP containing liposome treatment. Histologic examination of the CNS of liposome-treated animals confirmed decreased infiltration of macrophages into the parenchyma in the rats with T cell and AD-EAE, whereas T cells were still present.     

Suppression of acute and relapsing experimental allergic encephalomyelitis with mitoxantrone

The effect of treatment with the antineoplastic, immunomodulatory agent mitoxantrone on the course of acute and relapsing experimental allergic encephalomyelitis (EAE) in the mouse has been studied. Untreated mice immunized to produce acute EAE had an 81% incidence of clinical disease and 100% incidence of pathologic disease. Mice treated with mitoxantrone at a dose of 0.5 mg/kg daily for the 10 days following immunization did not develop any clinical signs and had minimal pathologic signs of disease. A dose of 0.25 mg/kg gave an intermediate response. Untreated mice immunized for relapsing EAE had a 100% incidence of disease with an average onset of disease on Day 148. Mice treated with mitoxantrone at a dose of 0.05 mg/kg three times weekly for 12 weeks following immunization had a 67% incidence of clinical disease with a significant delay in the average onset date to Day 279. These results indicate that mitoxantrone was highly effective in suppressing development of acute EAE. Mitoxantrone delayed the onset of relapsing EAE in mice, but did not fully inhibit the eventual expression of the disease. These studies suggest that the use of cytotoxic therapies in the treatment of autoimmune diseases may require periodic cycles of therapy to block disease expression.     

T suppressor hybridomas and interleukin-2-dependent lines induced by copolymer 1 or by spinal cord homogenate down-regulate experimental allergic encephalomyelitis

Suppressor T (Ts) hybridomas and interleukin-2-dependent T cell lines were established from spleens of mice, which had been rendered unresponsive to experimental allergic encephalomyelitis (EAE) either by mouse spinal cord homogenate or by the synthetic suppressant copolymer 1 (Cop 1). The Ts hybridoma supernatants and the Ts line cells specifically suppressed the in vitro response to the encephalitogenic myelin basic protein (BP), as indicated by inhibition of both the proliferation and interleukin-2-secretion responses of a BP-specific T cell line. Moreover, these Ts cells prevented the development of actively induced EAE in vivo. All hybridomas and lines were most effective when injected at the time of disease induction, thus suggesting that they operate as effector suppressor cells, and functionally inhibit encephalitogenic responses. The data presented here suggest that the suppressor cells are stimulated by the protective epitopes included in the BP as well as in the Cop 1 molecules and that they play an active role in the regulation of EAE. The generation of Ts lines and hybridomas, which have been induced by Cop 1, establish the specific stimulation of suppressor cells to EAE as a mechanism underlying the therapeutic activity of Cop l.     

Complement activation and expression during chronic relapsing experimental autoimmune encephalomyelitis in the Biozzi ABH mouse

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti‐myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1‐related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti‐complement agents might be of benefit in MS.     

Genetic influence on disease course and cytokine response in relapsing experimental allergic encephalomyelitis

A protracted and relapsing form of experimental allergic encephalomyelitis (EAE) develops in the DA rat after immunization with rat spinal cord homogenate (SCH) emulsified in incomplete Freund's adjuvant (IFA). The genetic influence on this model has been analyzed by immunizing MHC congenic strains on both LEW and DA genetic backgrounds, and recombinant inbred strains between DA and E3 rats. An in situ hybridization assay was used to examine the expression of mRNA for IFN-gamma, IL-4, IL-10 and transforming growth factor (TGF)-beta both in sections of spinal cords and the antigen-induced expression for these cytokines by splenocytes after in vitro stimulation with encephalitogenic MBP peptides. The susceptibility of relapsing EAE after immunization with SCH in IFA in the DA strain, but not the E3 strain, was correlated with a lack of expression for TGF-beta in the spinal cord. The recombinant inbred DXEB rats developed a severe EAE while surprisingly no signs of disease were observed in the DXEA strain, which shares the MHC region with the DXEB strain, after immunization with the MBP 63-87 peptide. Resistance to relapsing EAE in the DXEA strain correlated with increased non-MHC controlled expression for TGF-beta and lack of IFN-gamma in the spinal cord. The same pattern of cytokine expression was seen in splenocytes after stimulation in vitro with the MBP 63-87 peptide. A spreading of the immune response to the MBP 87-110 peptide was seen. Non-MHC genes controlled the quality of this response: splenocytes from MBP 63-87 immunized DXEB rats responded in vitro towards the MBP 87-110 peptide by expressing mRNA for IFN-gamma, IL-10 and IL-4, whereas in the DXEA strain the corresponding response involved IL-4 and TGF-beta. Taken together these data show that non-MHC controlled expression of mRNA for TGF-beta is associated with resistance to EAE.      

Induction of experimental allergic encephalomyelitis by myelin proteolipid-protein-specific T cell clones and synthetic peptides.

Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. SJL(H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 (HSLGKWLGHPDKF) develop acute experimental allergic encephalomyelitis (EAE). In the present study a T cell line and 4 clones were derived from SJL/J mice following immunization with this synthetic peptide. Severe clinical and histological EAE could be induced by adoptive transfer of the peptide-specific T cell line and 3 of 4 T cell clones. The T cell line/clones all responded strongly to PLP peptide 139-151 in in vitro proliferative assays. However, two different reactivity patterns emerged when truncated PLP peptides 141-150 and 141-149 were tested, suggesting that more than 1 epitope may be present within the PLP 139-151 determinant. To evaluate the encephalitogenic potential of the truncated peptides, we compared the ability of 2 truncated PLP peptides to induce EAE in vivo and proliferative responses in vitro. Immunization with PLP peptide 141-150 induced acute EAE in about 70% of mice tested, but PLP peptide 141-149 induced a comparatively mild form of EAE in 4 out of 9 mice tested. Lymph node cells from mice immunized with these peptides showed in vitro proliferative responses to each of the peptides, but the response to peptide 139-151 was always strongest. These combined in vivo and in vitro data further define the epitopes involved in PLP-induced EAE in SJL mice. Furthermore, the availability of multiple PLP-specific T cell clones will enable us to study the diversity of the T cell repertoire to PLP.     

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.     

T cell receptor antagonist peptides are highly effective inhibitors of experimental allergic encephalomyelitis

The feasibility of using T cell receptor (TcR) antagonist peptides to inhibit autoimmune disease has been examined. First, the fine antigenic structure of the I-A^s-restricted encephalitogenic determinant proteolipid protein (PLP) 139–151 has been analyzed. It was found that residues 145 and 148 were I-A^s anchor residues, and residue 144 appeared to be especially critical in T cell activation. Residues 142, 143, 146, and 147 were found to be crucial for activation of some, but not all, of the T cells studied. Next, good I-A^s-binding nonantigenic analogs were tested for TcR antagonism. Accordingly, several single substitution analogs were identified which could act as TcR antagonists. Moreover, when two such analogs were combined, the resulting TcR antagonist pool inhibited most of the PLP 139–151-specific T cell clones in vitro. When the efficacy of this TcR antagonist pool in inhibiting EAE induction in vivo was examined, it was found that the analog pool was a remarkably potent inhibitor of disease induction. The TcR antagonist pool was approximately 10-fold more potent than our best major histocompatibility complex blocker and was still capable of significant inhibition when injected in equimolar amounts with the encephalitogenic PLP 139–151 determinant.     

Recognition of alloantigens and induction of experimental allergic encephalomyelitis by a murine encephalitogenic T cell clone

In this communication we report a SJL/J (H-2s, Mlsc)-derived encephalitogenic T cell clone 4b.14a which has dual specificities for myelin basic protein/I-As and allogeneic H-2Ik gene products. Monoclonal antibodies specific for public class II major histocompatibility complex (MHC) determinants (Ia.17, I-Ak, r and Ia.7) and anti-L3T4 antibody inhibited the response of the clone 4b.14a to alloantigens, but a monoclonal antibody specific for a private determinant on I-Ak (Ia.2) did not inhibit the response. Although this clone proliferated in response to allogeneic spleen cells expressing H-2Ik determinants regardless of disparate Mlsa, b, c, d alleles, CBA/N cells (H-2k, Mlsnull) failed to stimulate the clone 4b.14a. These results suggest that recognition of allogeneic class II MHC molecules by this clone requires recognition with non-MHC gene products such as Mls. In addition, the clone 4b.14a stimulated by alloantigens could mediate clinical signs of experimental allergic encephalomyelitis in syngeneic recipients. However, interleukin 2 of rat spleen cell origin alone failed to activate the clone cells to make them encephalitogenic, though it could make them proliferate. The significance of these findings for T cell recognition and activation is discussed.     

T细胞定向迁移诱导实验性自身免疫性脑脊髓炎小鼠胸腺萎缩

目的 初步探讨实验性自身免疫性脑脊髓炎(EAE)小鼠胸腺萎缩的机制.方法 髓鞘少突胶质细胞糖蛋白(MOG)免疫C57BL/6小鼠诱导EAE,卵清白蛋白(OVA)免疫的小鼠作为对照;不同时间点计数胸腺、脾脏、淋巴结细胞总数,检测脾脏中胸腺来源细胞及中枢神经系统(CNS)浸润细胞.结果 MOG肽成功诱导EAE动物模型,小鼠出现典型的肢体运动功能障碍,脊髓可见大量炎性细胞浸润;MOG和OVA免疫均诱导胸腺细胞增加,第5天达到高峰,随后逐渐下降;EAE发病后胸腺细胞迅速减少,发病高峰期几乎完全消失,胸腺严重萎缩;MOG和OVA免疫后脾脏和淋巴结细胞总数持续升高,新近胸腺来源的T细胞增加尤其明显;EAE发病后脾脏T细胞总数减少,CNS浸润淋巴细胞总数增加.结论 大量T细胞在胸腺发育成熟并释放到外周,进而定向迁移至CNS诱导EAE是胸腺萎缩的主要原因.     

Modulation by cyclosporin-A of mononuclear cell distribution during experimental allergic encephalomyelitis

The capacity of CS-A to modify some inflammatory aspects of a cell mediated disease process, has been studied. In the present work, CS-A was shown to be effective at two levels during the development of experimental allergic encephalomyelitis (EAE) in rats. If CS-A was given at the time of the induction of the disease it inhibited lymphocyte proliferation in vivo, lymphocyte trapping into the draining lymph nodes, and delayed the subsequent infiltration of cells into the CNS and other inflammatory sites. When given around the time of disease manifestation, CS-A also reduced the rate of cell accumulation into the CNS and foot, but was without effect in modifying lymphocyte trapping in the draining nodes, despite the fact that this was still an ongoing process. Clinical signs were similarly delayed or reduced by both regimen of CS-A treatment. Treatment with CS-A did not lead, however, to long lasting unresponsiveness, since both treated groups suffered a relapse of disease at various times after treatment had been discontinued.