Susceptibility of Lewis Rats to Experimental Autoimmune Encephalomyelitis After Recovery from Passively Induced Disease

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by adoptive transfer of 5 × 10⁷ sensitized syngeneic lymphoid cells from donors challenged 12 to 43 days earlier with myelin basic protein and complete Freund's adjuvant. The donor cells were cultured for 72 hours with antigen prior to transfer. Following recovery, the recipients remained susceptible to EAE induced by challenge with antigen and adjuvant. The relevance of this finding to host recovery mechanisms is discussed.     

Regulation of experimental allergic encephalomyelitis III. Demonstration of effector cells in tolerant rats

Effector cells, which transfer experimental allergic encephalomyelitis (EAE), can be demonstrated in the spleens of Lewis rats which have been rendered tolerant to myelin basic protein (BP). Transfer of EAE was achieved following in vitro culture of tolerant donor spleen cells with antigen. The donor animals were, themselves, protected against EAE induced by active challenge with BP in complete Freund's adjuvant, although BP-activated effector spleen cells could partially override this protection. The finding that effector cells are present in unresponsive animals supports the hypothesis that immunologic self tolerance is actively regulated by suppressor mechanisms.     

Suppressor T cells prevent experimental autoimmune encephalomyelitis in mice.

Immune suppression (immunoprotection) in experimental autoimmune encephalomyelitis (EAE) was studied in (SJL X BALB/c)F1 mice using inocular of mouse spinal cord homogenate (MSCH), or mouse basic protein of myelin (M-BPM), in Freund's incomplete adjuvant (FIA). Such immunization specifically recruited lymphoid cells which markedly suppressed the capacity of effector lymph node cells from appropriately immunized syngeneic mice to transfer adoptively EAE. Suppression was demonstrable with transfer of bone marrow and spleen cells, but not with lymph nodes or thymus cells. Adoptively transferred suppression was maximal when cells were injected 9-30 days after the suppressive injection. Inhibition of EAE by suppressor cells was specific for the relavant antigen BPM, and required viable cells. Treatment of cells with anti-Thy-1 serum before transfer abolished their suppressor activity. After adoptive transfer of suppressor cells into syngeneic recipients subsequently immunized for EAE, there was inhibition of EAE and reduced cell-mediated immune response to BPM as judged by macrophage migration inhibition assays. Hence, in mice at least, immuno-protection against EAE is explicable by recruitment of suppressor T lymphocytes with the dual capacities of inhibiting development of effector T cells after antigenic stimulation, and of blocking their damaging effects on the antigen in the central nervous system.     

Enhancement of experimental autoimmune encephalomyelitis severity by ultrasound emulsification of antigen/adjuvant in distinct strains of mice

Susceptibility to experimental autoimmune encephalomyelitis (EAE) is associated with the major histocompatibility complex (MHC) haplotype. In this study EAE could be induced in six out of ten mice of the resistant DBA/2 (H-2^d) strain by ultrasound emulsified antigen/adjuvant, whereas none of the mice immunized with the conventional adjuvant developed the disease. Similar results were previously obtained for the MHC identical BALB/c mice. Further, while only few T cells were present in the central nervous systems (CNS) of the diseased DBA/2 mice, macrophages formed the majority of the infiltrates. In congenic BALB.B (H-2^b) and BALB.K (H-2 ^k) mice, EAE could be induced with both sonicated and extruded antigen/adjuvant emulsion. The results indicate that the EAE resistance in mice carrying the H-2^d MHC haplotype is dependent on the physical structure of the immunogen.     

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.     

The role of serum factors in the suppression of experimental allergic encephalomyelitis: evidence for immunoregulation by antibody to the encephalitogenic peptide.

Lewis rats immunized with myelin basic protein (MBP) in Freund's complete adjuvant (FCA) suffer from a single episode of paralysis from which they recover spontaneously. Subsequent to recovery, further episodes of paralysis cannot normally be induced by reimmunization with MBP in FCA. It is well established that serum, obtained from rats in the refractory state, can suppress the induction of experimental allergic encephalomyelitis (EAE) when given to animals from the time of immunization with MBP in FCA. Here it is shown that treatment with some such sera from Day 7 after immunization also suppressed the disease. However, not all convalescent sera were suppressive, indicating that rats immunized with MBP in FCA could become refractory to EAE without assayable levels of suppressive activity in their sera. In the context of this result it was notable that a correlation was found between the level of antibody specific for the encephalitogenic peptide in sera and the ability to suppress EAE. An inverse relationship was also shown between the amount of anti-encephalitogenic peptide antibody produced after immunization and the severity of EAE induced. Spleen cells from animals treated with Lewis anti-MBP serum after immunization with MBP in FCA could be activated to transfer EAE by in vitro culture with MBP despite the absence of any clinical signs in the donor animals, i.e. the serum inhibited the expansion or differentiation of these cells rather than preventing their priming or bringing about clonal deletion.     

Resistance to the induction of EAE in AO rats: its prevention by the pre-treatment with cyclophosphamide or low dose of irradiation

Susceptibility to the induction of EAE was compared in AO, DA and Lewis strain of rats. As evaluated by clinical and histological criteria, AO rats exhibited significantly lower susceptibility to EAE induced with guinea-pig spinal cord (GPSC) tissue and complete resistance to the encephalitogenic challenge with rat myelin basic protein (BP) irrespective of antigen dose and adjuvant used. AO rats pre-treated with BP + Freund's incomplete adjuvant became completely unresponsive to the induction of EAE with GPSC + Freund's complete adjuvant (FCA) indicating that they do possess cells sensitive to some antigenic determinants of rat BP. In order to test whether the resistance to EAE is due to an active suppression, low dose of irradiation (300 rad) and cyclophosphamide (20 mg/kg) was applied prior to the induction of EAE. Selective depletion of radiosensitive cells facilitated the induction of EAE. Similarly, cyclophosphamide given 2 days prior to BP + FCA completely abrogated the resistance to EAE induction. Thus, it appears that the inability of BP + FCA to produce EAE in AO rats is due to the disproportionate activation of suppressor cells.     

Autoimmune effector cells. II. Transfer of experimental allergic encephalomyelitis with a subset of T lymphocytes

This study was conducted to further characterize the effector cells of experimental allergic encephalomyelitis (EAE) which are activated in vitro when spleen cells from Lewis rats previously immunized with myelin basic protein and adjuvant are cultured with antigen prior to transfer to syngeneic recipients. The effector cells were isolated on discontinuous Percoll gradients in the cell fraction that floated on Percoll with a buoyant density of 1.067 kg/l. These cells (designated fraction 1) transferred EAE and incorporated [³H]dThd in culture. Fraction 1 was enriched for T cells when evaluated with monoclonal anti-rat T cell serum W3/13 and deficient in Ig⁺ cells; approximately 33% were positive with monoclonal anti-rat T cell serum W3/25. In contrast, the small, nonproliferating cells found in higher density Percoll fractions did not transfer EAE. When fraction 1 was recultured in the presence of basic protein and interleukin 2 for 72 h, these cells retained the ability to transfer EAE. Moreover, these recultured cells exhibited an increase in the W3/25 antigen and a decrease in the W3/13 marker. It was concluded that a subset of T cells which bear the W3/25 marker is involved in the transfer of EAE.     

Effect of indomethacin treatment upon actively-induced and transferred experimental allergic encephalomyelitis (EAE) in Lewis rats.

Daily administration of indomethacin (5 mg/kg) to Lewis rats from day 7 through day 21 following sensitization to guinea-pig spinal cord neural antigen plus Freund's complete adjuvant (FCA) resulted in an aggravation of clinical signs of EAE accompanied by a higher mortality. In contrast, lymph node cells (LNC) from sensitized and drug-treated donor rats showed decreased EAE transfer activity. The in vitro Con A response of LNC from sensitized, indomethacin-treated rats was comparable to those of normal LNC or LNC from sensitized animals only. The spleen cell response of sensitized rats was significantly lower than the response seen in normal animals, but was returned to normal levels in sensitized, indomethacin-treated animals. We believe these observations reflect an indomethacin-mediated diminished accumulation of EAE effector cells within peripheral lymph node tissues together with abrogation of splenic suppressor cell activity by indomethacin-mediated inhibition of prostaglandin (PG) synthesis in Lewis rats sensitized and treated with this drug under the conditions defined.     

The effect of cyclosporin A on the adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat.

Experimental allergic encephalomyelitis (EAE) can be adoptively transferred in Lewis rats with spleen cells from immunized animals, after culture with concanavalin A or myelin basic protein (MBP). The effect of the immunosuppressive drug cyclosporin A (CsA) on the in vitro and in vivo steps of the cell transfer has been investigated. Clinical signs of EAE were completely suppressed by CsA in rats immunized with MBP in Freund's complete adjuvant and spleen cells from these animals, cultured with the antigen, did not transfer the disease. The incidence of transferred disease was also reduced, if recipients were treated with CsA, although a higher dose of drug than that needed to suppress active EAE was required. In both instances complete suppression of EAE was only accomplished for the period of dosing, although the clinical signs of disease which appeared after withdrawal of the drug were significantly reduced in severity. These results imply that an immune response in the host animal is a prerequisite for adoptive transfer of EAE or suggest that CsA can regulate the action of lymphocytes already primed.     

Bone marrow-derived dendritic cells from experimental allergic encephalomyelitis induce immune tolerance to EAE in Lewis rats

We have previously shown that dendritic cells (DC), upon being pulsed in vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86) and injected subcutaneously (s.c.) back to healthy Lewis rats, transfer immune tolerance to experimental allergic encephalomyelitis (EAE) induced by immunization with MBP 68–86 and Freund's complete adjuvant (FCA). We here assumed that DC become pulsed in EAE rats, and that expansion in vitro of such ‘in vivo pulsed EAE‐DC’ might also have the capacity to induce immune tolerance to EAE, thereby eliminating the need for in vitro pulsing of DC with autoantigens which are still unknown in many autoimmune diseases in the human. In the present study, EAE‐DC were generated from bone marrow of Lewis rats, with EAE induced with MBP 68–86 + FCA, and expanded in vitro by culture with GM‐CSF and IL‐4. In comparison with DC from normal rats, EAE‐DC exhibited higher viability in the absence of growth factors, and presented specific antigen to naïve T cells in vitro. The DC derived from both EAE and healthy rats stimulated strong proliferation in an antigen‐independent manner, lasting for 4 weeks after DC were s.c. injected into healthy rats. During this time, injection of EAE‐DC did not induce clinical EAE. However, when these rats were immunized with MBP 68–86 + FCA, subsequent EAE was dramatically suppressed, and was associated with increased IFN‐γ expression, nitric oxide production, gradually reduced proliferation and cell apoptosis, compared with PBS‐injected control EAE rats. LPS‐treated DC did not induce tolerance, suggesting that the tolerance is mediated by an immature stage of DC. These observations support the hypothesis that EAE‐DC can transfer immune tolerance to EAE, thereby omitting the step of characterizing specific autoantigen. Omitting the step of loading DC with antigen not only eliminates the extremely complex procedure of defining pathogenically‐relevant autoantigens, but also avoids the risk of inducing immunogenicity of DC in the treatment of autoimmune diseases.     

Suppression of experimental allergic encephalomyelitis with thoracic duct lymphocytes

Thoracic duct lymphocytes (TDL) from Lewis rats immunized 9-10 days previously with basic protein in complete Freund's adjuvant (BP-CFA) failed to induce experimental allergic encephalomyelitis (EAE) in syngeneic recipients. This contrasts with the successful transfer of EAE by lymph node cell suspensions from donors immunized 9 days previously with BP-CFA. Only minor EAE was induced passively by TDL from rats immunized 11-12 days before with BP-CFA. TDL collected 9-20 days after BP-CFA immunization, however, were successful in transferring specific suppression of EAE tested by the lack of disease in the recipients immunized actively with BP-CFA 1 week after the TDL transfer. The data indicate that the thoracic duct contains specific suppressor cells shortly before, during and after the development of clinical EAE.     

The role of antigen absorption in the resistance of brown-Norway rats to experimental allergic encephalomyelitis

The designation of Brown-Norway (BN) rats as resistant to experimental allergic encephalomyelitis (EAE) is an oversimplification. Lewis rats are susceptible and BN rats are usually resistant to EAE after inoculation with guinea pig spinal cord or basic protein in Freund's adjuvant. However, EAE can be produced in BN rats by immunizing with rat cord and carbonyl iron, a particulate adjuvant. In the present work, the possibility that susceptibility of BN rats under these conditions is due only to the special qualities of the adjuvant has been eliminated by producing EAE in them without any adjuvant at all, merely by increasing the absorption and processing of the rat cord antigen. The susceptibility of the F1 hybrids is intermediate with respect to guinea pig cord antigen but it was equal to or greater than either parental strain when tested with rat cord antigen. Histologic evidence that BN rats do not absorb or process neural antigen as well as other strains, and the augmentation of EAE by increasing the dose and absorption of the inoculum, suggest that antigen absorption, processing and presentation is a "bottleneck" for development of EAE in BN rats. Absorption and processing of antigen should be considered along with cellular response, inflammatory mediators and epitope dominance when analyzing susceptibility and resistance to EAE.     

Antigen-specific splenic CD4+ and CD8+ regulatory T cells generated via the eye, suppress experimental autoimmune encephalomyelitis either at the priming or at the effector phase

The injection of antigen into the ocular anterior chamber (AC) induces the generation of splenic CD4(+) and CD8(+) regulatory T (Treg) cells, specific for the antigen injected into the AC. These Treg cells inhibit the induction (CD4(+)) and also the expression (CD8(+)) of a delayed-type hypersensitivity response. The ability of AC-induced self-antigen-specific Treg cells in modulating autoimmunity is not well defined. Here we show that an injection of encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55), suppresses the induction or progression of EAE, respectively. CD4(+) or CD8(+) splenic Treg cells induced by an injection of AC-MOG prevent EAE either at the inductive (priming) or at the progressive (effector) phase, respectively. This suppression of EAE by an AC-MOG injection or by intravenous transfer of splenic regulatory cells induced by an AC-MOG injection is specific for the antigen injected into the AC. Additionally, our data suggest that splenic CD8(+) Treg cells that suppress active EAE may use a transforming growth factor (TGF)-β-dependent suppression mechanism while the suppression of the induction of EAE by the AC-induced CD4(+) Treg cells is independent of TGF-β. Thus, we show for the first time that regulation of EAE at the priming or the chronic phase requires different phenotypes of Treg cells. Hence, it is important to consider the phenotype of Treg cells while designing effective cell-based therapies against autoimmune disorders.     

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

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

Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity

Summary: Experimental autoimmune encephalomyelitis (EAE) in the rat is an acute paralytic disease from which most animals spontaneously recover. The disease can be induced in susceptible inbred Lewis and DA rats with myelin basic protein (MBP), or encephalitogenic MBP peptides administered in complete Freund's adjuvant (CFA). The disease can be adoptively transferred to syngeneic recipients with primed T cells that have been reactivated in vitro with antigen. EAE is mediated by CD4⁺ Th1 cells that secrete proinflammatory cytokines, and spontaneous remission is associated with CD4⁺ T cells that secrete transforming growth factor-beta (TGF-β). Studies of EAE in susceptible rats have provided many important insights into the interactions of T cells and accessory cells that culminate in the induction of the autoimmune response.     

Induction of EAE by T cells specific for alpha B-crystallin depends on prior viral infection in the CNS

While myelin-reactive T cells are widely believed to play a pathogenic role in multiple sclerosis (MS), no substantial differences appear to exist in T-cell responses to myelin antigens between MS patients and healthy subjects. As an example, indistinguishable peripheral T-cell responses and serum antibody levels have been found in MS patients and healthy controls to alpha B-crystallin, a dominant antigen in MS-affected brain myelin. This suggests that additional factors are relevant in allowing myelin-reactive T cells to become pathogenic. In this study, we examined whether the inflammatory state of the CNS is relevant to the pathogenicity of alpha B-crystallin-specific T cells in mice. In normal mice, T-cell responses against alpha B-crystallin are limited by robust immunological tolerance. Reactive T cells were therefore generated in alpha B-crystallin-deficient mice, and these T cells were transferred into C57BL/6 recipients. While such a transfer in itself never induced any clinical signs of experimental autoimmune encephalomyelitis (EAE) in healthy recipient mice, acute EAE could be induced in animals that had been infected 7 days before with the avirulent A7(74) strain of Semliki Forest virus (SFV). SFV infection alone did not induce clinical disease, nor did it alter the expression levels of the target antigen. Our findings indicate that at least in mice, alpha B-crystallin-specific T cells can trigger EAE but only when prior viral infection has induced an inflammatory state in the CNS that helps recruit and activate T cells.     

Vaccination against experimental autoimmune encephalomyelitis using a subencephalitogenic dose of autoimmune effector cells (1). Characteristics of vaccination

We previously reported that rats could be vaccinated against EAE by inoculation with 10(7) anti-basic protein (anti-BP)-activated T cells raised as long-term lines. The activated T lines were irradiated (1,500 rads) to prevent them from causing EAE. We now report that a single inoculation of 10(4) or fewer cells of an activated anti-BP T-cell line did not cause clinical EAE but rather induced marked resistance to EAE produced by adoptive transfer of the anti-BP T cells. Resistance was less effective against EAE induced by active immunization to BP. Vaccination was immunologically specific, long lasting, and could be effected by various routes of administration.     

Two-step activation of T cells, clonal expansion and subsequent Th1 cytokine production, is essential for the development of clinical autoimmune encephalomyelitis

Lewis rats immunized with guinea pig myelin basic protein (GPBP) emulsified with incomplete Freund's adjuvant (IFA) do not develop experimental autoimmune encephalomyelitis (EAE). However, we found that GPBP/IFA with pertussis toxin (PT) administration induced full-blown EAE. By comparing the immunological status of rats immunized with GPBP/IFA plus PT [PT (+) rats] with that of rats immunized with GPBP/IFA alone [PT (-) rats], we tried to elucidate the pathomechanisms of EAE. Analysis of the TCR clonality by CDR3 spectratyping revealed that Vbeta8.2 and Vbeta10 expansion of T cells occurred in both PT (-) and PT (+) rats, indicating that activation of T cells at this level is not sufficient for the development of clinical EAE. Quantitation of cytokine mRNA and protein revealed that PT (-) rats showed a Th2-dominant, while PT (+) rats showed a Th1-dominant, cytokine profile. Furthermore, administration of IL-12, but not of IFN-gamma and TNF-alpha, induced clinical EAE in GPBP/IFA-immunized animals. Taken together, two-step activation, activation of T cells bearing a particular type of TCR by antigen immunization and subsequent overproduction of Th1 cytokines, mainly IL-12 production, induced by appropriate adjuvants is essential for the development of clinical EAE.     

Acquired thymic tolerance and experimental allergic encephalomyelitis in the rat. I. Parameters and analysis of possible mechanisms

Intrathymic injection of guinea pig myelin basic protein (MBP) or the immunodominant, ence phalitogenic fragment of MPB, 68 – 86, without otherwise compromising the peripheral lymphocyte pool in adult LEW rats, dramatically inhibits onset of experimental allergic encephalomyelitis (EAE) caused by the usual peripheral inoculation with MBP in complete Freund's adjuvant. This surprising finding demonstrates that interaction of antigen and one or more components of an intact thymus can down-regulate systemic responses by mature T cells already existing in the peripheral lymphocyte pool. How this happens is not known. In studies designed to explore possible mechanisms: (a) adult thymectomized animals remain susceptible to active EAE, thus EAE cannot be attributed solely to recent thymic emigrants that might be inactivated by antigen deposited in the thymus; (b) heterotopic isografts of injected thymic lobes transfer thymic tolerance to secondary recipients, thus the tolerance effect is dominant over an intact, non-treated thymus; (c) T cells from made thymic tolerant but not immunized donors are less effective in causing EAE following adoptive transfer into, and active immunization of, secondary, irradiated recipients; and (d) animals resistant to active EAE as a consequence of thymic tolerance are fully vulnerable to adoptive EAE caused by already activated MBP-specific T cell subpopulations. These results rule out a possible mechanism previously proposed for acquired thymic tolerance, i. e., that potentially pathogenic T cells traffic to the antigen-injected thymus where they are inactivated or eliminated.