Suppression of experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein

The oral administration of myelin basic protein (MBP) to Lewis rats prior to an encephalitogenic challenge resulted in total inhibition or a significant delay in the onset of experimental autoimmune encephalomyelitis (EAE). In vitro lymphocyte proliferative responses to MBP were significantly decreased in MBP-fed rats when compared with vehicle-fed controls. Suppression of EAE and in vitro proliferative responses to MBP were observed to be antigen specific, since oral feeding of a control protein exerted no suppressive effect. Moreover, the specificity of MBP-induced oral tolerance was shown to be species specific, since feeding guinea pig MBP (GPMBP) or human MBP (HuMBP) induced protection only against a GPMBP or HuMBP challenge, respectively. Conversely, Lewis rats could not be orally tolerized to the self antigen rat MBP.     

THE ENCEPHALITOGENIC ACTIVITY AND MYELIN BASIC PROTEIN CONTENT OF ISOLATED OLIGODENDROGLIA

Abstract— Oligodendroglia isolated from frozen human brain induced EAE in the guinea pig when injected with Freund's complete adjuvant. An acid extract of the isolated cells was highly encephalitogenic. The disease was clinically and histologically similar to EAE induced with CNS white matter or myelin basic protein(MBP). The isolated cells were essentially myelin-free but contained a large amount of myelin basic protein as determined by polyacrylamide gel electrophoresis. It was shown that the cells can absorb ¹²⁵I-MBP during isolation and the possibility that contamination from extra cellular MBP is responsible for the encephalitogenic activity is considered.     

Competition between foreign and self proteins in antigen presentation. Ovalbumin can inhibit activation of myelin basic protein-specific T cells.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurological disease initiated by activated T cells specific for the autoantigen, myelin basic protein (MBP). The ability of Lewis rat splenic T cells to transfer EAE after in vitro incubation with MBP-pulsed dendritic cells (DC) was used as an index of MBP-specific T cell activation. OVA, previously processed by macrophages, was incubated with MBP and DC at the pulsing stage to determine whether it could inhibit presentation of the autoantigen. At molar equivalents of 2.5:1 and 20:1 relative to MBP, processed OVA increasingly inhibited the ability of DC to activate MBP-specific T cells for EAE transfer. Unprocessed OVA, which cannot be presented immunogenically by Lewis rat DC, was much less effective. However, processed OVA added to DC after they had been pulsed with MBP could not compete. OVA also blocked appearance of EAE when mixed with MBP/CFA in the inoculum used for active induction of the disease. Splenic T cells from MBP + OVA/CFA-immunized rats transferred EAE with a substantially delayed onset, suggesting that a reduced number of MBP-specific T cells was generated by immunizing with the OVA + MBP mixture compared with MBP alone. Overall, the data indicate that fragments of a foreign protein, OVA, which can be bound by APC, can also inhibit presentation of encephalitogenic determinants of MBP to T cells.     

Antigen processing of myelin basic protein is required prior to recognition by T cells inducing EAE.

The processing and presentation of whole myelin basic protein (MBP) and a 12 amino acid encephalitogenic peptide were investigated using MBP-immune and peptide-immune murine T cell lines. Myelin basic protein is the major component of central nervous system (CNS) white matter capable of inciting an autoimmune response which leads to the disease, experimental allergic encephalomyelitis (EAE), in a number of animal species. MBP-immune T cell lines caused a form of adoptively transferred EAE when injected into naive, syngeneic recipients. It has been found that both whole MBP and peptide required processing in order to induce proliferation of the T cell lines. The proliferative response was greatest when MBP was processed under conditions in which proteolysis was prevented. The demonstration that activation of encephalitogenic MBP immune T cells requires a processed form of MBP may have relevance to the human inflammatory CNS demyelinating condition, multiple sclerosis, for which EAE is the EAE is the prime animal model.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Characterization of the antigen specificity and TCR repertoire,and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.     

Encephalitogenic and proliferative responses of Lewis rat lymphocytes distinguished by position 75- and 80-substituted peptides of myelin basic protein

The encephalitogenic and proliferative responses of Lewis rat lymphocytes were defined by use of synthetic peptide GP68-84, representing the 68-84 sequence of guinea pig myelin basic protein (GPMBP), and otherwise identical peptides containing substitutions of either A75 or P80 residues. The comparative activities of these peptides were tested in the following bioassays: 1) active induction of experimental autoimmune encephalomyelitis (EAE), 2) potentiation of EAE transfer activity by MBP- or peptide-sensitized lymph node cells (LNC), 3) in vitro proliferation of MBP- or peptide-sensitized LNC, and 4) in vitro proliferation of an encephalitogenic T cell line. The GP68-84 peptide exhibited potent activity in all four bioassays. In contrast, [A75]GP68-84 and [P80]GP68-84 exhibited a selective loss of certain activities while retaining activity in other bioassays. For example, LNC were activated by culture with [A75]GP68-84 to express potentiated EAE transfer activity. Furthermore, [A75]GP68-84 and GP68-84 were equipotent in stimulating the proliferation of the encephalitogenic T cell line. However, [A75]GP68-84 was virtually inactive in assays measuring the induction of EAE or the proliferation of either GPMBP- or [A75]GP68-84-sensitized LNC. Conversely, the [P80]GP68-84 peptide actively induced EAE and potentiated EAE cellular transfer activity but was incapable of stimulating proliferation of either GPMBP-sensitized LNC or an encephalitogenic T cell line. When [P80]GP68-84 was used for sensitization, in vitro proliferation of LNC was stimulated, but only by MBP sequences containing a P80 substitution. Overall, these results indicate that at least two structurally distinct T cell determinants of GP68-84 regulate functionally diverse encephalitogenic and proliferative activities of EAE-associated T cells.     

Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects

Experimental allergic encephalomyelitis (EAE)-susceptible Lew and EAE-resistant Brown Norway (BN) rats and the corresponding MHC congenic strains were examined for their ability to develop clinical and histologic EAE. The ability of T cells from these animals to proliferate in vitro in response to whole guinea pig (GP) myelin basic protein (MBP), rat MBP, and to the major encephalitogenic peptide of GP MBP 66-88 (GP 68-88) was also assessed. We found that Lewis (Lew) was highly susceptible and showed good T cell responses to GP, MBP, rat MBP, and GP 68-88. Lew.1N (BN MHC on Lew background) and BN were not susceptible and T cells from these strains showed significant responses to GP MBP, but not to rat MBP or GP 68-88. Although BN.B1 (Lew MHC on BN background) was not susceptible to actively induced EAE, MBP-specific Lew T cells could transfer severe disease to BN.B1. BN.B1 T cells showed responses to GP-MBP, rat MBP, and GP 68-88 and, when transferred to naive BN.B1 or Lew, induced only mild clinical EAE in both strains. Increasing the number of T cells from BN.B1 had no effect on the severity of clinical symptoms in either recipient, suggesting some deficiency in the T cell repertoire that is necessary for induction of severe EAE. These results suggest that 1) the T cell response to rat MBP and GP68-88 (but not to sites other than 68-88 in GP MBP) is necessary for susceptibility to EAE; 2) the ability to respond to both rat MBP and GP 68-88 is determined by the MHC gene products on APC; and 3) given a permissive MHC, the T cell response that results in EAE is influenced by non-MHC genes.     

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.     

T cell specificity for class II (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic protein

The role of class II restriction in T cell recognition of an epitope of the autoantigen myelin basic protein (MBP) has been investigated. Encephalitogenic PL/J(H-2u) and (PL/J X SJL/J(H-2s))F1 ((PLSJ)F1) clones, isolated after immunization with intact MBP, recognize the N-terminal 11 amino acid residues of MBP in association with I-Au class II molecules. The synthetic peptide MBP 1-11 has been tested in vivo for induction of EAE. Clinical and histological EAE occurs in PL/J and (PLSJ)F1 mice but not SJL/J. The class II restriction of T cells primed with MBP 1-11 has been examined in primary cultures in vitro. Similar to encephalitogenic T cell clones, isolated after continuous selection in vitro, the population of MBP 1-11-specific proliferative PL/J and (PLSJ)F1 T cells, recognize this epitope in association with I-Au class II molecules. Not all MBP-specific T cell clones which are restricted to I-Au class II molecules cause autoimmune encephalomyelitis. The specificity of these non-encephalitogenic clones has been examined in this report. These clones also recognize MBP 1-11. Thus recognition of an encephalitogenic T cell epitope is not sufficient for induction of EAE.     

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments

We report that experimental autoimmune encephalomyelitis, a T cell-mediated autoimmune disease studied as a model for multiple sclerosis, can be suppressed in Lewis rats by the oral administration of myelin basic protein (MBP). Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner. In addition, proliferative responses to MBP and, to a lesser extent, serum levels of anti-MBP antibody were suppressed by feeding MBP. Suppression of clinical and histologic disease was observed whether animals were fed MBP before or after disease induction, although suppression was more complete when rats were fed before immunization. Disease was also suppressed by the oral administration of either encephalitogenic or nonencephalitogenic fragments and decapeptides of the MBP molecule, with more complete suppression observed when nonencephalitogenic fragments were fed, suggesting that suppressor determinants exist in the MBP molecule distinct from the encephalitogenic region. The oral administration of a non-disease-inducing portion of an autoantigen represents an antigen-specific method by which an experimental autoimmune disease can be immunoregulated.     

Measurement of cell-mediated inflammation in experimental murine autoimmune encephalomyelitis by radioisotopic labeling.

A radioisotopic index test was used to detect that time of onset and intensity of cell-mediated immune inflammation of experimental autoimmune encephalomyelitis (EAE) in mice. Mice were tested at various time intervals after an encephalitogenic immunization with mouse spinal cord to homogenate for delayed-type hypersensitivity (DTH) to myelin basic protein (MBP) by intradermal challenge with antigen in the ear pinna. After 25 hr, the intensity of DTH was measured by 125I-radiometry which depends upon the migration of 125I-UdR radiolabeled mononuclear cells into the antigen depot. Cells reactive to MBP were detected by the ear assay as early as 7 days after the initial encephalitogenic sensitization. The degree of cell-mediated immune inflammation in the brain and spinal cord during the evolution of EAE was also measured by a radioisotopic technique; increased 125I-UdR uptake could be detected in the brain 3 to 4 days before the onset of signs of EAE at days 11 to 12, whereas 125I-UdR in the spinal cord was detected only 1 day before, or concomitant with, the onset of signs of EAE. Both, or concomitant with, the onset of signs of EAE. Both the "ear" and "organ" radiometric index tests are useful in measuring the degree of cell-mediated inflammation in EAE, and supplement routine histopathological and observational assessments.     

Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein epitope 72-85 with a human MBP(87-99) analogue and effects of cyclic peptides

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). In the present report, a linear analogue and a series of cyclic semi-mimetic peptides were designed and synthesized based on the human myelin basic protein (MBP(87-99)) epitope (Val87-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro90) and on Copolymer I (a mixture of random polymers of Ala, Gln, Lys and Tyr used to treat MS). These analogues were designed looking for suppressors of EAE induced by guinea pig MBP(72-85) epitope (Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro-Val) in Lewis rats. The linear analogue [Arg91,Ala96]MBP(87-99), in which Arg substitutes Lys91 and Ala substitutes Pro96, was found to be a strong inhibitor which when administered to Lewis rats together with the encephalitogenic agonist MBP(72-85) completely prevented the induction of EAE. In contrast, three N- and C-termini amide-linked cyclic semi-mimetic peptides, [cyclo-Phe-Arg-Asn-Ile-Val-Thr-Ala-Acp (1), cyclo-Phe-Ala-Arg-Gln-Acp (2), cyclo-Tyr-Ala-Lys-Gln-Acp (3)] as well as a Lys side chain and C-terminous cyclic semi mimetic peptide cyclo(Lys, Acp)-Phe-Lys-Asn-Ile-Val-Thr-Ala-Acp (4) which contain segments of MBP(87-99) or are constituted from immunophoric residues of copolymer 1, were ineffective in inducing or inhibiting EAE in Lewis rats. However co-injection of cyclic analogues with MBP(72-85) delayed the onset of EAE indicating a modulatory effect on the EAE activity of MBP(72-85). These findings suggest that molecule length, size of cyclic moiety and backbone conformation are important elements for immunogenic activity. Moreover blockade of MBP(72-85) induced EAE by the unrelated peptide [Arg91,Ala56]MBP(87-99) could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.     

A rabbit B cell determinant for a conserved portion of myelin basic protein, rabbit encephalitogenic sequence 65-74

Synthetic peptide S24 (TTHYGSLPQKG) represents residues 65-74 of myelin basic protein (MBP) and contains the major determinant involved in the development of experimental allergic encephalomyelitis (EAE) in rabbits. This peptide is completely conserved in all nonprimate mammals for which sequence information is available. Although it is clear that peptides containing the S24 region are capable of inducing EAE, previous serologic studies have resulted in the conclusion that the determinant is "buried" or sequestered in intact MBP. Employing a liquid phase radioimmunoassay, we studied Ab responses to the S24 determinant in six rabbits injected with rat myelin. Two of the six animals developed small but measurable responses to the S24 determinant. In one of these rabbits, the response was boosted with a covalent conjugate of S82 and methylated BSA (MBSA). We also measured antibodies to the S24 determinant in rabbit antisera to human, monkey, dog, bovine, and the large and small forms of rat MBP. By nonequilibrium inhibition analysis, we determined that the antibody responses to these antigens were all directed to a determinant composed of residues 66-71 of MBP, and that intact MBP inhibits the binding of these antibodies to radiolabeled S24. The results demonstrate that the rabbit encephalitogenic region of myelin basic protein is exposed in the intact molecule both as an immunogen and as a reactant in liquid-phase assays; furthermore, they demonstrate that MBP antigenicity leading to B cell responses does not necessarily depend on sequence differences between the injected protein and its counterpart in the host species. The latter finding reinforces the contention of Atassi that autoantibody responses are not exclusive to "evolutionary hypervariable locations."     

Mast cell proteases liberate stable encephalitogenic fragments from intact myelin.

Protease-containing supernatants from activated rat mast cells were found to degrade purified rat myelin with a subsequent release of a stable encephalitogenic peptide. The two most abundant peptides were identified as residues 69-87 (GSLPQKSQRTQDENPVV) and residues 69-88 (GSLPQKSQRTQDENPVVH). While additional exposure to the mast cell supernatants removes the COOH terminal histamine from peptide 69-88 to yield peptide 69-87, additional proteolytic degradation of the 69-87 peptide was not detected. Immunization with this peptide emulsified in CFA caused the development of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In addition this 69-87 sequence was found to activate resting encephalitogenic myelin basic protein-reactive T cell lines to adoptively transfer clinical EAE. The release of stable encephalitogenic peptides from the myelin sheath by mast cell proteases may play a role in activation of encephalitogen-specific T cells during the progression of EAE.     

Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis.

Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.     

Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype

T helper cells reactive to myelin basic protein are clearly implicated in the pathogenesis of murine EAE. We have developed a T cell line, BML-1 that (1) is reactive to the encephalitogenic amino terminal nonapeptide (1-9NAC) of MBP, (2) is I-Au restricted, and (3) induces relapsing EAE in B10.PL (H-2u) mice. Measurement of the lymphokine profile of BML-1 revealed secretion of IL-2, interferon-gamma and lymphotoxin but not IL-4. This profile is consistent with the Th1/DTH subtype. Coculture of BML-1 with MBP-primed B cells shows that BML-1 does not provide significant helper function in vitro. In addition, BML-1 secretion of interferon-gamma was found to inhibit LPS-induced anti-MBP antibody responses. This suggested that anti-MBP antibodies may not be necessary for induction of EAE. Sera from mice, in which severe disease was induced with the 1-9NAC peptide and Bordetella pertussis, showed no development of serum antibodies to MBP. These data show that MBP-reactive Th cells of the Th-1/DTH subtype can induce EAE and do not provide Th function for anti-MBP responses and that serum anti-MBP antibodies are not found in peptide 1-9NAC-induced disease. T cell lines specific for encephalitogenic epitopes and characterized for lymphokine secretion will provide a useful tool for understanding the role of T cells in the induction of EAE.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system

Multiple sclerosis (MS) is a devastating neuroinflammatory disorder of the central nervous system (CNS) in which T cells that are reactive with major components of myelin sheaths have a central role. The receptor for advanced glycation end products (RAGE) is present on T cells, mononuclear phagocytes and endothelium. Its pro-inflammatory ligands, S100-calgranulins, are upregulated in MS and in the related rodent model, experimental autoimmune encephalomyelitis (EAE). Blockade of RAGE suppressed EAE when disease was induced by myelin basic protein (MBP) peptide or encephalitogenic T cells, or when EAE occurred spontaneously in T-cell receptor (TCR)-transgenic mice devoid of endogenous TCR-alpha and TCR-beta chains. Inhibition of RAGE markedly decreased infiltration of the CNS by immune and inflammatory cells. Transgenic mice with targeted overexpression of dominant-negative RAGE in CD4+ T cells were resistant to MBP-induced EAE. These data reinforce the importance of RAGE-ligand interactions in modulating properties of CD4+ T cells that infiltrate the CNS.