Experimental allergic encephalitis: study of cellular immunity to the encephalitogenic determinant.

Guinea pigs were tested for cellular immunity to the encephalitogenic tryptophan peptide, the major encephalitogenic determinant of central nervous system basic protein, representing residues 114 to 122 of the molecule. Guinea pigs sensitized with human basic protein regularly developed experimental allergic encephalitis, but did not show cellular immunity to the encephalitogenic tryptophan peptide as measured by skin test reactivity, lymphocyte stimulation, or macrophage migration inhibition, although they did show cellular immunity to the immunizing antigen, human basic protein. Animals sensitized with the synthetic tryptophan peptide also regularly develop clinical and histologic features of experimental allergic encephalitis, and show cellular immunity to the peptide but not to human basic protein. The work of others indicates that, in guinea pigs sensitized with the whole basic protein, there are determinants for cellular immunity located near the encephalitogenic tryptophan peptide. The test peptides used in these studies all included amino acid residues of the basic protein not included in the encephalitogenic tryptophan peptide used in our study. Our work indicates that the encephalitogenic peptide is not one of the determinants for cellular immunity in the basic protein molecule. Since cellular immunity to the disease-producing determinant of the molecule could not be demonstrated, this work further suggests that cellular immunity, as measured by the three tests described herein, may not necessarily be correlated with production of experimental allergic encephalitis.     

Serum degradation of myelin basic protein with loss of encephalitogenic activity: evidence for an enzymatic process.

Fibrin deposition in parallel with loss of myelin basic protein (MBP), an antigenic constituent of central nervous system (CNS) myelin, within the lesions of animals with experimental allergic encephalomyelitis (EAE) suggested that degradation of MBP by proteolytic activity associated with blood clotting might be an important immunopathologic event in this prototypic autoimmune disease. Following incubation in normal rat serum at 37 °C for more than 4 hr, but not to any comparable degree in plasma, MBP had little or no encephalitogenic activity when bioassayed in guinea pigs or rats. Fragments of increasingly lower molecular weight were demonstrable by polyacrylamide gel electrophoresis after addition of MBP to rat serum; no fragments appeared after incubating the protein in rat plasma. Little or no loss of encephalitogenic activity was observed when MBP was incubated in serum containing protease inhibitors. These findings indicate that the serum-mediated degradation of MBP and concomitant loss of encephalitogenic activity is due to an enzymatic process associated with the coagulation cascade or/and the complement, kallikrein or fibrinolytic pathways. Implications of these findings concerning EAE and the multiple sclerosis process in man are discussed.     

Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.     

Correlation of cytotoxicity with experimental allergic encephalomyelitis.

Cytotoxicity of immune lymph node cells in experimental allergic encephalomyelitis (EAE) was maximal 9 days after injection of encephalitogenic emulsion. The ability of these cells to passively transfer EAE was also maximal at this time. Immune spleen cells were more cytotoxic than lymph node cells 9 days after injection; however, these cells did not passively transfer EAE. Twelve days after injection of encephalitogenic emulsion immune spleen cells passively transferred EAE with resulting mild histopathologic lesions. At this time the spleen cells were 50% more cytotoxic than comparable lymph node cells. Cyclophosphamide suppressed the development of clinical EAE and the development of cytotoxic lymphoid cells. It also reduced clinical signs and cytotoxic activity of lymph node cells. Spleen cell cytotoxic activity was enhanced by Cyclophosphamide. It was concluded that cytotoxic activity of lymph node and spleen cells was correlated with the ability of these cells to produce EAE. Lymph node cell populations differed qualitatively and/or quantitatively from immune spleen cell populations in EAE. Capacity to passively transfer EAE coincided with the maximal Cytotoxicity of the lymphoid cells from each tissue.     

Experimental allergic encephalomyelitis. Isolation of basic proteins and polypeptides from central nervous tissue

1. Basic protein (mol.wt. 16500) and polypeptides (mol.wt. 3500) were isolated from bovine spinal cord by a procedure involving defatting, acid extraction of the defatted material and repeated chromatography on Sephadex G-50. Similar fractions were isolated from guinea-pig brain. 2. These fractions produced experimental allergic encephalomyelitis in guinea pigs. 3. The polypeptides appeared to be derived from a basic protein of myelin as a result of the action of an acid proteinase during extraction with acid. Similar proteolysis might also occur in the isolation of other biologically active polypeptides from acetone-dried powders of nervous tissue. The activity of the acid proteinase was lowered by defatting with chloroform-methanol. 4. Peptides from tryptic digests of encephalitogenic polypeptides and protein were also encephalitogenic, which suggests that the encephalitogenic determinant may be quite a short sequence of amino acids. 5. These encephalitogenic polypeptides are further examples of antigens of low molecular weight.     

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 acquisition of encephalitogenicity after sensitized cells are conditioned with myelin basic protein or concanavalin A

When cells from rats immunized against neural antigens are incubated with myelin basic protein or concanavalin A, their ability to transfer experimental allergic encephalomyelitis (EAE) is greatly increased. Using the rapid, localized form of EAE, we have shown that the increased encephalitogenicity of these cells was not manifest, or not fully manifest, in 1 day after passive transfer. Expression of the increased encephalitogenic potency required a period of residence in the recipient animal and this period did not depend on the duration of incubation in vitro. Conditioned cells were fully capable of eliciting the neutrophilic form of EAE, which is consistent with the notion that the recipient's mononuclear cells were not essential for the expression of the encephalitogenic potential of conditioned donor cells.     

Modulation of adoptive cell transfers in experimental allergic encephalomyelitis by antigen treatment of donors

Experimental allergic encephalomyelitis has been adoptively transferred using lymph node cells from Strain 13 guinea pig donors sensitized with purified encephalitogenic myelin basic protein. Adoptive cell transfer was used to examine the immunocompetence of lymph node cells obtained from guinea pigs protected from disease development by treatment with MBP. Lymph node cells from guinea pigs unresponsive to EAE challenge do not adoptively transfer disease. Cells obtained from guinea pigs treated with MBP following encephalitogenic challenge are competent in adoptive transfer with respect to pathologic lesions, but not clinical disease. The clinical and pathologic responses of recipients of the histocompatible lymphocyte populations are similar to those seen in the treatment-matched donor controls, suggesting that under these circumstances lymphoid cells, rather than circulating soluble factors, are responsible for disease induction and suppression.     

The isolation and properties of experimental allergic encephalitogenic protein

1. Experimental allergic encephalitogenic (EAE) protein was isolated from ox spinal cord by a modification of the method of Martenson & LeBaron (1966). 2. The protein was examined by acrylamide-gel electrophoresis and its amino acid composition determined. 3. Sedimentation-velocity runs in the ultracentrifuge indicate a molecular weight of about 15100 at pH7.8, and calculation suggests that approx. 137 amino acid residues are present per molecule. 4. Gel-filtration and diffusion studies suggest that the protein is non-globular. 5. Optical-rotatory-dispersion measurements show the protein to have no helical secondary structure even at pH values near to the isoelectric point. In the presence of triphosphoinositide, changes in the optical rotatory dispersion of the protein could be interpreted to mean that it develops a small degree of secondary structure. 6. On treatment with cyanogen bromide the experimental allergic encephalitogenic protein is split into at least two fragments, the larger of which is only about 12% smaller than the parent protein and is antigenically active, and the smaller of which is devoid of antigenic activity.     

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.     

The presence of specific antigen-reactive cells during the induction, recovery, and resistance phases of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is an induced disorder in which an autoimmune response specific for myelin basic protein (BP) results in neural tissue destruction and acute paralysis. Lewis rats rapidly recover from induced paralysis and do not display any clinical manifestations of EAE following a second BP injection. The object of this study was to determine if immunologic recognition of encephalitogenic fragments of the BP molecule occurs during the induction, recovery, and resistance phases of EAE. Paralysis was induced in Lewis rats by a single injection of BP in complete Freund's adjuvant (CFA). The results indicate that macrophage migration inhibition in the presence of encephalitogenic BP fragments containing amino acid residues 43–88 and 68–88 is detectable during the paralytic stage of EAE, and also following recovery from clinical neurologic impairment. Although rats which had recovered were resistant to secondary BP-induced paralysis, macrophage migration inhibition in the presence of BP, or its encephalitogenic fragments, was detected after the second BP-CFA challenge. In order to assess humoral immunologic recognition, levels of serum antibody specific for the 43–88 BP fragment were determined. Specific antibody was not detected during the paralytic episode, but appeared upon recovery. Specific antibody was also detected following the secondary BP-CFA challenge. These data indicate that antigen-sensitive cells are present during the induction, recovery, and resistance phases of EAE in the Lewis rat. The mechanism which controls the activity of these cells in vivo has not been established.     

Antigen-stimulated rosette formation by T lymphocytes in experimental allergic encephalomyelitis

Peripheral blood lymphocytes form rosettes in the presence of heterologous etythrocytes. Spontaneous or active rosette formation has been reported to be a measure of circulating and immunologically functional thymus-dependent lymphocytes. The present study utilizes the rosette assay to measure changes in the circulating T cells of guinea pigs sensitized with encephalitogenic myelin basic protein (BP) or with nonencephalitogenic peptide S42 known to induce cellular transformation in experimental allergic encephalomyelitis, a cell-mediated disorder of the central nervous system. The results show a significant depression in the number of active but not in the total number of rosette-forming T lymphocytes from the peripheral blood of antigen-sensitized animals. This reduction, which was not related to the encephalitogenic property of the BP, was readiiy reversible by incubating lymphocytes with the sensitizing antigen but not with histone. Under these conditions, lymphocytes from unsensitized control animals were unresponsive to stimulation by any of the antigens used. The antigenstimulated rosette assay described in this report provides a specific assay for sensitization to basic protein in BP-related demyelinating diseases.     

Theoretical conformational analysis of a encephalitogenic peptide molecule and a study of the structure-activity relationship in a series of its analogs

Semi-empirical energy calculations are used to determine all low-energy conformations of Trp-containing fragment 113-121 of myelin basic protein (experimental allergic encephalomyelitis inducing peptide). The computed conformations are compared with the results of physico-chemical experiments and data on biological testing of the encephalitogenic peptide analogs. The three computed structures are shown to be in a good agreement with the available experimental evidence. However, additional information is required to predict "biologically active" conformation of encephalitogenic peptide.     

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.     

Experimental allergic encephalomyelitis: basic protein regions responsible for delayed hypersensitivity

Three separate peptide regions were isolated from the chymotrypsin digest of the encephalitogenic basic protein from bovine myelin of the central nervous system. The peptides induced delayed type hypersensitivity (DTH) and elicited delayed skin reactivity in experimental animals. However, none of the isolated peptides was capable of inducing experimental allergic encephalomyelitis (EAE). The amino acid sequence of peptide CTP-3 (Gly-Ala-Glu-Gly-Gln-Lys-Pro-Gly-Phe-OH) and peptide CTP-la were found to overlap the C-terminal sequence of encephalitogenic peptides E (residue 112–125) and T8 (residue 65–74) of the basic protein, respectively. The third DTH inducing peptide, CB1-T1, (N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH) was found to overlap the N-terminal sequence of the basic protein molecule. Common to the three DTH inducing peptides, to the basic protein and to the encephalitogenic peptides E-S and T8S is the X-X-X-Gln-Lys sequence. Isolation of the regions of the basic protein that are responsible for DTH provides antigens for the study of the mechanism of cellular immunity in EAE.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant

Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freund's complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node.The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.     

Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice

C57BL/6 mice immunized with the extracellular Ig-like domain of rat myelin oligodendrocyte glycoprotein (MOG) developed experimental autoimmune encephalomyelitis (EAE) resembling that induced by rodent MOG 35-55 in its B cell independence and predominantly mononuclear CNS infiltrate. In contrast, human MOG protein-induced EAE was B cell dependent with polymorphonuclear leukocytes. Human MOG differs from rat MOG at several residues, including a proline for serine substitution at position 42. Human MOG 35-55 was only weakly encephalitogenic, and a proline substitution in rat MOG at position 42 severely attenuated its encephalitogenicity. However, human MOG 35-55 was immunogenic, inducing proliferation and IFN-gamma and IL-13 to human, but not rodent MOG 35-55 [corrected]. The B cell dependence of EAE induced by human MOG protein was not due to a requirement for Ag presentation by B cells, because spleen cells from B cell-deficient mice processed and presented human and rat MOG proteins to T cells. The different pathogenic mechanisms of human and rat MOG proteins might result from different Abs induced by these proteins. However, rat and human MOG proteins induced Abs to mouse MOG that were equivalent in titer and IgG subclass. These data demonstrate that EAE can be induced in C57BL/6 mice by two mechanisms, depending on the nature of the immunogen: an encephalitogenic T cell response to rat MOG or rodent MOG 35-55, or an encephalitogenic B cell response to epitopes on human MOG protein that most likely cross-react with mouse determinants.     

On basic proteins in bovine peripheral nerve myelin.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.     

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.