Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein

Experimental allergic encephalomyelitis (EAE), an experimental autoimmune disease of the central nervous system (CNS), is readily induced in many mammalian species by immunization with CNS tissue or myelin basic protein (MBP) purified from the CNS. EAE has been frequently used as a model for multiple sclerosis (MS). However, EAE generally presents as an acute monophasic disease in the adult animal after immunization with MBP. After recovery, the animal is resistant to rechallenge with encephalitogen (1). Two exceptions to these observations have been reported. McFarlin et al. (2) reported that a variable number of Lewis rats showed signs of a single, mild relapse about a week after recovery from MBP-induced acute EAE. Panitch and Ciccone (3) have reported induction of recurrent EAE in rats immunized with human MBP. Chronic, relapsing EAE has been induced in the mouse; however, an apparent requirement for CNS tissue had been noted (4, 5). Recently, during the course of a series of experiments on the induction of EAE in SJL/J, PL/J, and (SJL/J X PL/J)F1 (SPL F1) mice, it was observed that the F1 mice frequently had paralytic relapses after recovery from MBP-induced symptoms. Experiments were initiated to examine this phenomenon, and the findings are presented below.     

Failure of myelin basic protein to prevent or suppress experimental allergic encephalomyelitis in guinea pigs

The encephalitogenic myelin basic protein (BP) was reported to be effective in preventing and suppressing the development of experimental allergic encephalomyelitis (EAE) when animals were treated before or after encephalitogenic challenge, respectively. In this report we show that pretreatment with 15 daily doses of 2.5 or 0.15 mg homologous BP (in IFA) failed to protect guinea pigs from subsequent challenge with encephalitogenic emulsion. Similarly, 15 daily injections of 1.0, 2.5, 5.0, or 10.0 mg guinea pig BP (in IFA) did not suppress development of or arrest ongoing EAE when the treatment was initiated on days 1, 4, 8, or 11 after an encephalitogenic challenge. The results show that over 50% of the treated animals developed hind leg paralysis (HLP), incontinence, or both, and the incidence of HLP was not altered significantly by a 10-fold increase in the amount of BP used for daily treatment. Further, all the treated and challenged animals developed histological lesions characteristic of disease. Treatment with BP delayed disease onset, prolonged the period of paralysis leading to recovery from HLP, and reduced both the prevelence of histological lesions as well as the incidence of death. It may be concluded that under these experimental conditions the administration of BP failed to protect from or suppress development of EAE.     

The suppression of experimental allergic encephalomyelitis in Lewis rats by treatment with myelin basic protein-cell conjugates

Pretreatment of Lewis rats with guinea pig (GP) myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) suppressed the subsequent induction of experimental allegic encephalomyelitis (EAE) with GP-MBP in Freund's complete adjuvant. The degree of suppression correlated positively with the amount of antigen coupled to the SL. GP-MBP coupled to syngeneic red blood cells (RBC) also resulted in suppression of EAE and the extent of the suppression was related to the dose of cells. These regimens of pretreatment also resulted in a decrease in the in vitro lymphocyte proliferative response to GP-MBP and in the extent of perivascular cuffing in the spinal cord. No decrease in the anti-MBP antibody response was detected in rats pretreated with either GP-MBP-SL or GP-MBP-RBC conjugates. Transfer of lymph node cells from rats pretreated with GP-MBP-RBC resulted in a decrease in disease severity in recipients. It is concluded that prior administration of MBP-cell conjugates is an effective way of suppressing the symptoms of EAE.     

The development of experimental allergic encephalomyelitis with immunizing doses of myelin basic protein.

Rabbits immunized with low (11.25 mg) and high (57.50 mg) doses of myelin basic protein from several species develop antibasic protein antibodies, delayed type hypersensitivity, and clinical and pathological signs of experimental allergic encephalomyelitis. More than 55% of rabbits immunized with relatively high doses of basic protein develop disease. The absence of circulating antibasic protein antibodies in immunorespondent animals is associated with the appearance of clinical or histological signs of experimental allergic encephalomyelitis; however, the presence of humoral antibodies did not prevent completely the development of disease. Delayed-type hypersensitivity, specific for the basic protein, appears as early as 5 days after immunization and is maintained in nondiseased and surviving animals. Neither excess encephalitogen nor encephalitogen-induced antibody is sufficient to suppress completely the eventual development of clinical or histological manifestations of disease.     

Studies on the mechanism of suppression of experimental allergic encephalomyelitis induced by myelin basic protein-cell conjugates

The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the disease-inducing injection of MBP in Freund's complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells.     

Successful immunization against experimental allergic encephalomyelitis with myelin basic protein-sensitized allogeneic lymphocytes

Prevention and suppression of experimental allergic encephalomyelitis were demonstrated in rats, guinea pigs, and rabbits immunized with allogeneic, but not with syngeneic lymphocytes from susceptible donors sensitized to myelin basic protein (MBP). Donor lymphnode, splenic, or peripheral blood lymphocytes were effective in inducing a state of unresponsiveness to an encephalitogenic challenge in either of the three species. Unresponsiveness was not obtained in recipients immunized with sensitized allogenic lymphocytes and simulatenously challenged with MBP suggesting that a time lapse between immunization and challenge is necessary for the development of protective immunity. Induced in immunized recipients, unresponsiveness was transferred into normal syngeneic recipients with immunoglobulin-G (IgG) isolated from protected donors before challenge. Furthermore, both immunized and IgG recipients failed to develop cell-mediated immunity after challenge with MBP. The results show that prevention and suppression of EAE was mediated by antibodies which inhibited the development of delayed type hypersensitivity to the challenging antigen.     

Cell-mediated immunity in experimental allergic encephalomyelitis: cross reactivity between myelin basic protein and mycobacteria antigens.

Guinea pigs injected with Freund's incomplete adjuvant emulsified with guinea pig spinal cord, purified guinea pig myelin basic protein, or human myelin basic protein showed dermal reactivity to both of the basic proteins as well as to mycobacteria antigens. Animals receiving only mycobacteria antigens expressed dermal reactivity to the sensitizing antigen in addition to basic protein. This cross reactivity may help explain the role of mycobacteria in inducing and protecting against EAE, and may have important implications concerning human demyelinating diseases.     

Covalent linkage of phosphoinositides to myelin basic protein: in vivo occurrence and in vitro studies with experimental allergic encephalomyelitis

We have previously reported the covalent attachment of phosphoinositides to myelin basic protein (MBP) in vitro. In this study, in vivo phosphoinositidation of MBP was observed to occur after intracerebral injection with [32p]HPO4(2-). In the in vitro study of experimental allergic encephalomyelitis, a four-fold increase in phosphoinositidation of MBP was observed in the myelin from the guinea pigs sacrificed during the development of motor dysfunction, as compared to the control group. A decrease (40%) was observed among most of the animals sacrificed before the onset of motor dysfunction.     

Oral administration of myelin basic protein is superior to myelin in suppressing established relapsing experimental autoimmune encephalomyelitis

Oral administration of a myelin component, myelin basic protein (MBP), induces immunological unresponsiveness to CNS Ags and ameliorates murine relapsing experimental autoimmune encephalomyelitis (REAE). However, a recent clinical trial in which multiple sclerosis patients were treated with repeated doses of oral myelin was unsuccessful in reducing disease exacerbations. Therefore, we directly compared the tolerizing capacity of myelin vs MBP during REAE in B10.PL mice. Oral administration of high doses of myelin, either before disease induction or during REAE, did not provide protection from disease or decrease in vitro T cell responses. In contrast, repeated oral administration of high doses of MBP suppressed established disease and MBP-specific T cell proliferation and cytokine responses. The frequency of IL-2-, IFN-gamma-, and IL-5-secreting MBP-specific T cells declined with MBP feeding, implicating anergy and/or deletion as the mechanism(s) of oral tolerance after high Ag doses. We have previously shown that the dosage and timing of Ag administration are critical parameters in oral tolerance induction. Studies presented here demonstrate that Ag homogeneity is also important, i.e., homogeneous Ag (MBP) is more effective at inducing oral tolerance than heterogeneous Ag (myelin).     

A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.     

Delayed-type hypersensitivity to myelin basic proteins in mice susceptible to allergic encephalomyelitis

The delayed-type hypersensitivity (DTH) response in mice immunized with autologous spinal cord homogenate or purified myelin basic protein (MBP) was measured by the¹²⁵I-UdR uptake ear assay. Mice were tested for DTH responses with MBP preparations from different species and with synthetic peptides. The 114–122 and 68–84 peptide regions appear to be major determinants for inducing and eliciting DTH in the mice which are susceptible to allergic encephalomyelitis.Special Issue dedicated to Dr. Elizabeth Robez-Einstein.This investigation was supported by Grant No. 1256-B-3 and RG 1197-B7 from the National Multiple Sclerosis Society, N.Y. and a grant from The Margaret T. Biddle Foundation. D.S.L. is a Scholar of the Leukemia Society of America, Inc.     

Subtle effects on myelin basic protein-specific T cell responses can lead to a major reduction in disease susceptibility in experimental allergic encephalomyelitis

The presence of potentially autoreactive T cells is a necessary, but not sufficient, condition for the development of autoimmune disease. However, the relationship between T cell response and susceptibility to disease is not straightforward. In this report, we use experimental allergic encephalomyelitis as a model to demonstrate that subtle alterations of the T cell response to an encephalitogenic epitope are sufficient to cause a dramatic decrease in disease susceptibility. Transgenic expression of a fusion protein of hen egg lysozyme and an encephalitogenic peptide of myelin basic protein (MBP) residues 84-105, coexpressed with MHC class II, causes profound tolerance to hen egg lysozyme, while maintaining a near normal response to MBP. Detailed analysis of the T cell repertoire of transgenic animals using a panel of T cell hybridomas revealed a highly selective loss of one minor component of the response to the MBP84-104 region. Despite this, transgenic animals were highly resistant to experimental allergic encephalomyelitis induction with the MBP peptide, indicating that minor changes to the T cell repertoire may result in major alterations in disease susceptibility. Possible reasons for this are discussed.     

Indomethacin augments in vitro proliferative responses of Lewis rat lymphocytes to myelin basic protein. Implications for experimental autoimmune encephalomyelitis

Indomethacin (IM), a specific inhibitor of prostaglandin (PG) synthesis, and PGE2 were studied in terms of their ability to modulate in vitro immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Lymphoid cells from either the spleens or the draining lymph nodes of myelin basic protein (MBP)-sensitized rats exhibited in vitro immune responses which were enhanced in the presence of IM. Specifically, IM enhanced (i) guinea pig MBP (GPMBP)- and rat MBP (RMBP)-stimulated lymphocyte proliferation, (ii) background proliferation, and (iii) interleukin 2 (IL-2)-stimulated proliferation. Conversely, PGE2 inhibited both GPMBP- and IL-2-stimulated proliferation of MBP-sensitized lymphocytes. Together, these results indicate that PGs secreted by cultured lymphoid cells can directly mitigate MBP- or IL-2-stimulated lymphocyte proliferation. Furthermore, the observation that IM and PGE2 modulate in vitro responses of MBP-specific lymphocytes may provide insight into how the in vivo administration of IM potentiates the severity of EAE (H. Ovadia and P.Y. Paterson, Clin. Exp. Immunol. 49, 386, 1982) and how PGs may be involved in the spontaneous remission of EAE in rats.     

Experimental allergic encephalomyelitis: sequestered encephalitogenic determinant in the bovine myelin basic protein.

The presence of a sequestered encephalitogenic determinant for Lewis rats in the bovine myelin BP was demonstrated with synthetic peptide sequences prepared in our laboratory by the Merrifield solidphase method. The sequence of the encephalitogenic determinant (residues 75-84) from bovine BP (peptide S6), H-Ala-Gln-Gly-His-Arg-Pro-Gln-Asp-Glu-Asn-OH, is similar but not identical to the sequence reported for the guinea pig BP (peptide S53), H-Ser-Gln-(–)-(–)-Arg-Ser-Gln-Asp-Glu-Asn-OH. The presence or the absence of Gly-His from the sequence of either the bovine or the guinea-pig determinants did not alter their encephalitogenic potencies; however, the presence of Gly-His at positions 77 and 78 together with H-Gly-Ser-Leu-Pro-Gln-Lys- (residues 69-74) at the N-terminal end of the bovine determinant destroyed its encephalitogenic potency. In contrast to the absence of Gly-His from the potent encephalitogenic guinea-pig BP, guinea-pig fragment 44-89, and synthetic peptide S49, its presence in the bovine sequence prevents recognition of this determinant and renders the parent bovine BP, bovine fragment 44-89, and synthetic peptide S8 (residues 69-84) relatively non-encephalitogenic. The results of this study suggest that intramolecular interactions occur between adjacent amino acids, conferring secondary or tertiary structures upon this region of the bovine BP which renders the encephalitogenic determinant inaccessible for recognition by the host animal. The presence of sequestered disease-inducing determinants needs to be considered in choosing a particular BP for therapeutic use.