Potassium Channel Blockers Inhibit Adoptive Transfer of Experimental Allergic Encephalomyelitis by Myelin-Basic-Protein-Stimulated Rat T Lymphocytes.

Agents which block T cell K(+) currents can prohibit both proliferative and effector cell functions in T cells activated by mitogens or phorbol esters. This study examined the effects of some of these blocking agents on the immune responsiveness of guinea pig myelin basic protein (GPMBP)-reactive Lewis rat T lymphocytes, which are capable of mediating the adoptive transfer of experimental allergic encephalomyelitis (EAE), an accepted animal model for multiple sclerosis. Both the proliferative functions (DNA synthesis and cell blastogenesis) and the EAE transfer activities of GPMBP-reactive lymphocytes were examined following GPMBP-induced activation in the presence of agents shown to block the outwardly rectifying K(+) current in these cells. At concentrations which completely inhibited DNA synthesis, as measured by [(3)H]thymidine incorporation, and cell blastogenesis, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and methoxyverapamil (D60) completely blocked the subsequent adoptive transfer of EAE into naive syngeneic Lewis rats. The concentrations at which these blockers produced a 50% reduction in DNA synthesis were estimated to be 16, 1.6 and 32 &mgr;M for TEA, 4-AP and D-600, respectively, which were roughly equivalent to the EC(50) to block the K(+) current. Apamine, a potent Ca(2+)-activated K(+) channel blocker, at a concentration several orders of magnitude higher than is necessary to block Ca(2+)-activated K(+) channels, reduced the maximal K(+) conductance in GPMBP-reactive T cell K(+) channels by about 20%, but did not alter either [H(3)H]thymidine incorporation or the adoptive transfer of EAE. These results indicate that delayed rectifier K(+) channel blockers may prevent the activation of GPMBP-reactive T cells, thus prohibiting encephalitogenic effector cell functions. Copyright 1997 S. Karger AG, Basel

Modulation of Outward K(+) Conductance Is a Post-Activational Event in Rat T Lymphocytes Responsible for the Adoptive Transfer of Experimental Allergic Encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an accepted animal model for the human demyelinating disease multiple sclerosis. The continuously propagated line of Lewis rat T helper lymphocytes (GP1 T cells), specific for the encephalitogenic 68-86 sequence of guinea pig myelin basic protein (GPMBP), mediates the adoptive transfer of EAE into normal syngeneic Lewis rats. Because mitogenic activation of T cells can increase K(+) conductance, this study investigated changes in the outwardly rectifying K(+) conductance in GP1 T cells following activation with the encephalitogen, GPMBP. Using the gigohm.seal whole-cell variation of the patch clamp technique, GP1 T cells were studied during a 3-day culture with GPMBP and throughout the subsequent 10 days, as cells progressed through both GPMBP-induced activation (EAE transfer activity) and proliferation responses, finally reverting to the resting state. Resting GP1 T cells exhibited peak K(+) conductances around 2 nS, while GPMBP-induced activation resulted in 5- to 10-fold increases in peak K(+) conductance, which temporally coincided with the optimal period for EAE transfer activity. During and immediately after the optimal period for EAE transfer, 20-mV depolarizing shifts in the voltage dependence of both activation and inactivation developed, abruptly reversing to resting values as cells reverted to the resting state. Accompanying the depolarizing shifts were a slowing of the K(+) current activation kinetics and an acceleration of the deactivation kinetics. These results indicate that the K(+) conductance in GP1 rat T helper cells is modulated over the full time course of GPMBP-induced cellular responses and that K(+) channels should be optimally available during the period of adoptive EAE transfer, preceding disease manifestation. Copyright 1997 S. Karger AG, Basel

Differential recognition of sequences within the encephalitogenic region of myelin basic protein capable of eliciting cell-mediated immune responses in experimental autoimmune encephalomyelitis.

The fine specificity of myelin basic protein (MBP) epitopes capable of eliciting in vivo delayed-type hypersensitivity responses in Lewis rats with experimental autoimmune encephalomyelitis (EAE) was compared to those eliciting in vitro antigen-specific T cell proliferation and augmentation of disease transfer. Utilizing a panel of synthetic peptides with sequences representing the 68-86 region of guinea pig (GP-) or bovine myelin basic protein (B-MBP), animals were primed with one species of peptide and subsequently challenged with either the same peptide or peptides with truncations or substitutions representative of the other species of MBP. In regard to minimal length sequences capable of eliciting delayed-type hypersensitivity (DTH), rats primed with GP-MBP and complete Freund's adjuvant (CFA) exhibited a hierarchical pattern of responsiveness to challenge with a series of truncated peptides, ranking as follows: GP-68-86 > GP-72-86 > GP-68-84 > > GP-75-86 = no activity. This response pattern corresponds to that previously reported for T cell proliferation and activation for disease transfer. Furthermore, a comparison of these T cell-mediated immune parameters, as elicited by the substituted peptides, revealed the response patterns of DTH reactivity to be similar to that previously described for in vitro T cell proliferation with significant DTH responses generated only by the peptide species for which the animal was primed. In contrast, a cross-reactive pattern of recognition was observed in cells mediating disease transfer, with all four 68-86 sequences capable of augmenting activation for adoptive transfer of disease, regardless of the peptide species for which the animal was primed.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurovascular permeability and fibrin deposition in the central neuraxis of Lewis rats with cell-transferred experimental allergic encephalomyelitis in relationship to clinical and histopathological features of the disease.

Abrupt increases in blood-brain barrier (BBB) permeability were detected by the dual-isotope technique, coinciding with evidence of activation of coagulation cascade, occurred 1 day prior to appearance of clinical neurological signs of experimental allergic encephalomyelitis (EAE) and in conjunction with initial detectable cell infiltration. Maximal increase of BBB permeability was observed on the first day of clinical signs, which was 2 days prior to maximum severity of clinical abnormalities and 1 day in advance of the greatest number of central nervous system (CNS) fibrin deposits and perivascular cellular infiltration. Returning of increased BBB permeability and CNS perivascular fibrin deposits to normal levels was demonstrated prior to complete remission of neurological signs. Considerable CNS perivascular cellular infiltrates, however, lasted after complete remission of neurological signs. These findings indicate that increased permeability of the BBB, in association with activation of the coagulation cascade, is the earliest expressions of immune effector activity of experimental allergic EAE.

Concordance and localization of maximal vascular permeability change and fibrin deposition in the central neuraxis of Lewis rats with cell-transferred experimental allergic encephalomyelitis.

This study utilized Lewis rats and the cell-transfer form of experimental allergic encephalomyelitis (EAE) to focus on two central nervous system microvascular alterations known to be intimately associated with early clinical neurological signs of the disease, namely increased vascular permeability and deposition of fibrin. The main objective of the work was to define the degree of concordance and the anatomical localization of maximal vascular permeability change and fibrin deposition within the neuraxis of recipient rats during the earliest expression of clinical manifestations of the disease. Recipients were injected with predetermined doses of activated, myelin basic protein (MBP)-reactive syngeneic donor lymphoid cells and killed 6 or 7 days later when they exhibited the first clinical signs of disease. This experimental design allowed assessment of late occurring immune activities of a single population of MBP-reactive effector cells. Increased vascular permeability, expressed as mean extravascular blood equivalents (EVBE), attained maximum change, i.e., 16.04 +/- 5.07, within the lumbosacrococcygeal segment of the spinal cord (LSC). Maximal density of fibrin depositions, i.e., 192 +/- 94 deposits, also occurred within the LSC cord. The concordance of these two histoimmunopathological events, their tight linkage to early clinical signs of disease and the fact that both events attained maximal values within the LSC cord suggest that increased permeability and activation of the coagulation cascade may be prerequisite events for the expression of neurological signs of EAE.

Antigen-specific inhibition of the adoptive transfer of experimental autoimmune encephalomyelitis in Lewis rats.

The efficacy of antigen-specific immunoregulation as a treatment for the efferent limb of an autoimmune disease was tested in a rat model of adoptive experimental autoimmune encephalomyelitis (EAE). Lewis rats receiving 4-5 x 10(7) guinea pig (GP) myelin basic protein (MBP)-activated lymph node T cell blasts from GPMBP/CFA sensitized donors routinely show clinical signs of disease 5-6 days post transfer. Intravenous injection of GPMBP coupled to syngeneic splenocytes using the chemical cross-linker carbodiimide was effective in completely abrogating the expression of clinical EAE in rats that received MBP-specific T cells 2 days previously. Partial inhibition was also observed in rats injected as early as day 0 (the same day as MBP-specific T cell transfer) and as late as 1 day prior to the onset of clinical signs (days 4-5 post transfer). Unresponsiveness was shown to be dose-dependent, dependent on the route of injection of the neuroantigen-coupled splenocytes, and was antigen-specific. Splenocytes coupled with GP or rat MBP (which are identical within the major encephalitogenic GP68-86 Lewis rat determinant with the exception of the residue at position 80) were equally efficient at eliminating disease expression in recipients of GPMBP-specific T cells. In contrast, splenocytes coupled with bovine or rabbit MBP (which differ significantly from GPMBP within the 68-86 region) had no inhibitory effect. The antigen specificity of the tolerance induction was also illustrated by the fact that splenocytes coupled with GP68-86, but not those coupled with the truncated GP68-84 peptide, induced profound unresponsiveness. Interestingly, de novo antigen processing by the antigen-coupled cells did not appear to be necessary as the inclusion of antigen processing inhibitors had no effect on inhibition of disease. However, the use of the carbodiimide coupling reagent was critical for the induction of unresponsiveness as essentially equivalent amounts of 125I-labelled MBP were bound in its presence or absence, but only splenocytes incubated in the presence of both MBP and carbodiimide inhibited clinical expression of disease. Antigen-specific tolerance is thus an effective means of inhibiting expression of clinical disease in the rat EAE model, and a powerful tool for determining the fine epitope specificity of encephalitogenic T cells.

Neurovascular fibrinolytic activity in normal Lewis rats and rats with cell-transferred experimental allergic encephalomyelitis.

Fibrinolytic activity in the form of plasminogen activator (PA) was assessed using a histochemical fibrin slide technique in spinal cords of normal Lewis rats and rats with the cell-transferred form of experimental allergic encephalomyelitis (EAE). PA was localized exclusively to blood vessels. Vessels in the leptomeninges had maximum activity. A precipitous decrease in PA activity occurred in recipient rats which coincided with onset of clinical neurologic signs. A subsequent return in activity occurred in association with clinical remission of disease but remained well below the activity level of normal rats for as long as the recipient animals were followed. Vessels containing perivascular cellular infiltrates of EAE had little or no detectable PA activity. Furthermore, PA could not be demonstrated to be associated with infiltrating inflammatory cells, including macrophages. These findings provide further support for involvement of the coagulation and fibrinolytic systems in the early clinical manifestations of EAE in Lewis rats.

Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease.

The N- and C-terminal boundaries of myelin basic protein determinants required for encephalitogenic and proliferative responses of Lewis rat T cells.

Highly purified synthetic peptides representing portions of the 68-86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to define the N- and C-termini of encephalitogenic determinants that cause experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Each peptide was tested for: (a) induction of EAE, (b)in vitro potentiation of EAE transfer activity by GPMBP-sensitized lymph node cells (LNC), (c) in vitro proliferation of GPMBP-sensitized LNC, and (d) in vitro proliferation of a GPMBP-reactive line of EAE-inducing T cells. In these bioassays, the general rank order of potency was: GPMBP greater than or equal to GP68-86 greater than or equal to GP72-86 greater than [G84]GP68-86 greater than or equal to GP68-84 much greater than GP75-85 greater than or equal to GP75-84 = virtually no activity. These results demonstrate that the encephalitogenic region is bounded by the 72-74 and 84-86 sequences. Further evidence presented herein indicates that the 75-84 sequence contains the primary antigenic features required for specific T cell recognition of the encephalitogenic region.

Distinct accessory cell requirements define two types of rat T cell hybridomas specific for unique determinants in the encephalitogenic 68-86 region of myelin basic protein.

Six clonotypically unique T cell hybridomas from Lewis rats were used to study accessory cell activities required for class II MHC restricted T cell responses to the 68-86 encephalitogenic sequence of myelin basic protein (MBP). T cell hybrids which were cultured with GP68-86 68-86 sequence of guinea pig MBP (GPMBP) and naive splenocytes (SPL) were induced to produce IL-2 as measured by the CTLL indicator cell line. The hybrids were categorized into two subsets (designated THYB-1 and THYB-2), because two distinct subset-specific pathways of communication between accessory cells and T cells were involved in GPMBP-induced IL-2 production. These pathways were distinguished by the following six observations. First, when the duration of a pulse of SPL with GPMBP was lengthened from 1 to 4 h, these SPL lost their ability to induce IL-2 production by THYB-2 hybrids yet nevertheless retained full stimulatory activity for THYB-1 hybrids. Second, paraformaldehyde fixation of GPMBP-pulsed SPL abrogated an activity necessary for Ag-induced IL-2 production by THYB-2 hybrids. These fixed SPL were nevertheless able to stimulate THYB-1 hybrids, albeit to a lesser extent than viable unfixed SPL. Third, the addition of either cycloheximide, cytochalasin B, or 2-deoxyglucose to an Ag pulse of SPL with GPMBP dramatically inhibited the subsequent responses of THYB-2 hybrids yet had little or no effect upon the reactivity of THYB-1 hybrids. Fourth, thymocytes lacked necessary activities for GPMBP evoked IL-2 production by THYB-2 hybrids yet strongly promoted THYB-1 hybrid responses. Fifth, exposure of SPL to as little as 500 rad of gamma-irradiation markedly attenuated THYB-2 hybrid response to GPMBP but did not affect THYB-1 responses. Sixth, anti-GPMBP responses by THYB-2 hybrids were observed only in the presence of both radioresistant adherent SPL and a distinct population of radiosensitive nonadherent SPL. Conversely, THYB-1 hybrids exhibited full reactivity to GPMBP in the presence of adherent radioresistant SPL. Together, these observations reveal that two distinct accessory cell-T cell pathways mediate immune recognition of the 68-86 encephalitogenic region of MBP. Furthermore, these results indicate that subsets of Th cells can be defined by the accessory cell type-specific interactions that are necessary for Ag-mediated responses.

Clonotypic heterogeneity of Lewis rat T cells specific for the encephalitogenic 68-86 region of myelin basic protein.

Experimental autoimmune encephalomyelitis was induced in a Lewis rat by sensitization with synthetic peptide GP68-86, representing the 68-86 sequence of guinea pig myelin basic protein (GPMBP). To delineate T cell determinants of GP68-86, lymph node cells from this rat were activated in culture with GP68-86 and were fused with cells of the mouse thymoma BW5147. The resultant hybrids were cloned by limiting dilution and screened for GP68-86-evoked secretion of IL2 in the presence of rat splenocytes. Twelve T cell hybrids derived in this manner were tested for reactivity to different heterologous species of MBP as well as to substituted or truncated analogs of GP68-86. The hybrids generally exhibited potent reactivity to GPMBP but differed markedly in their reactivity to autologous rat MBP (RMBP). A few exceptional hybrids exhibited crossreactivity with peptides in which native serine75 or serine80 residues of GPMBP were substituted with either alanine75 (A75) or proline80 (P80) residues. These cross-reactive hybrids also possessed high levels of anti-RMBP reactivity. The remaining hybrids were unresponsive to the A75 and P80 substituted peptides and, with one exception, had relatively low levels of anti-RMBP reactivity. Unique reactivity patterns were also revealed by hybrid responses to peptides having modified C-terminal 84-86 residues. In summary, the contrasting fine specificities of different hybrids indicated that several distinct clones of T cells mediate the immune response of Lewis rats against the 68-86 region of GPMBP. Furthermore, heterogeneity in the hybrid response to "self" RMBP may reflect substantial differences in encephalitogenic potency of the T cell clones from which these hybrids were derived.

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.

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.

Interleukin 1 and myelin basic protein synergistically augment adoptive transfer activity of lymphocytes mediating experimental autoimmune encephalomyelitis in Lewis rats.

This investigation focused on the role of adherent accessory cells and their cellular product, interleukin 1 (IL 1), in cellular immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Guinea pig myelin basic protein (GPMBP)-sensitized lymph node cells (LNC) responded in culture with GPMBP by undergoing activation as measured by augmented transfer of EAE to syngeneic recipients, and proliferation as measured by [3H]thymidine incorporation. GPMBP-sensitized LNC, after depletion of adherent accessory cells, no longer responded to GPMBP in the EAE transfer activation assay. In contrast, aliquots of the same LNC preparation exhibited proliferative responses to GPMBP that were only partially reduced. Addition of irradiated thymocytes to adherent cell-depleted cultures fully reconstituted responsiveness to GPMBP in the activation assay and restored full reactivity to GPMBP in the proliferation assay. Furthermore, addition of either purified human IL 1 or recombinant human IL 1 to adherent cell-depleted cultures reconstituted reactivity to GPMBP in the EAE transfer activation assay and augmented GPMBP-specific proliferative responses. Anti-Ia monoclonal antibodies blocked GPMBP + IL 1-induced cellular activation of nonadherent LNC. These results demonstrate that both IL 1 and Ia molecules are important in the pathway leading to GPMBP-induced activation of EAE-inducing T lymphocytes. Furthermore, these results suggest that different accessory signals may be required for optimal induction of GPMBP-induced lymphocyte activation vs GPMBP-specific proliferative responses.

Suppression of clinical signs of cell-transferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with a plasminogen activator inhibitor.

The purpose of this study was to determine whether fibrinolysis resulting from activation of the clotting cascade in juxtaposition to endothelial cells of the central nervous system (CNS) microvasculature is important for development of clinical signs of experimental allergic encephalomyelitis (EAE) in recipient Lewis rats. Rats were injected with previously primed syngeneic lymph node cells, activated in vitro with guinea pig myelin basic protein, and subsequently treated daily with trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCA), a synthetic inhibitor of plasminogen activator. Clinical signs of EAE were significantly suppressed in AMCA-treated rats compared to saline-treated control recipient animals. Furthermore, suppression of clinical signs in AMCA-treated rats was accompanied by a significant curtailment in EAE-associated increased permeability of the blood-brain barrier (BBB). These findings provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of the BBB, are related prerequisite events for expression of clinical manifestations of EAE.

Role of the clotting system in the pathogenesis of neuroimmunologic disease.

Experimental allergic encephalomyelitis (EAE) is a prototypic neuroautoimmune disease involving sensitization to central nervous system myelin basic protein (MBP). Our studies of the clotting system and ensuing fibrinolysis implicate coagulation and cleavage of fibrin within or on the luminal surface of the cerebrovasculature as events initiating the inflammation characterizing EAE. Among recipient rats injected with MPB-primed, cultured-activated lymph node cells, opening of the blood-brain barrier (BBB) and deposition of perivascular fibrin within the spinal cord occur in parallel 1 day before onset of clinical signs of EAE. Daily treatment of recipient rats with trans-4-(aminomethyl)cyclohexanecarboxylic acid, a synthetic product that specifically inhibits plasminogen activator derived from endothelial cells, results in marked reduction of increased permeability of the BBB and suppression of clinical signs of EAE. We postulate that the critical event precipitating EAE is binding of circulating MBP-reactive immune effector cells to MBP immunodeterminants on the surface of cerebrovascular endothelial cells. Coagulation and ensuing fibrinolysis occur at sites of binding of effector cells to cerebrovascular endothelium. Release of biologically active peptides cleaved from fibrin open the BBB, thereby setting the stage for the cascade of inflammatory events culminating in clinical manifestations of EAE.

Induction of experimental allergic encephalomyelitis in Lewis rats with purified synthetic peptides: delineation of antigenic determinants for encephalitogenicity, in vitro activation of cellular transfer, and proliferation of lymphocytes.

Four highly purified synthetic peptides encompassing segments of the 68-86 region [for the numbering system used, see Eylar, E.H., Brostoff, S., Hashim, G., Caccam, J. & Burnett, P. (1971) J. Biol. Chem. 246, 5770-5784] of myelin basic protein (MBP), a region known to induce experimental allergic encephalomyelitis (EAE) in Lewis rats, were used to define and compare structure-function relationships between the primary structure of the 68-86 sequence and the three following biological activities: induction of EAE in Lewis rats, stimulation of T lymphocytes in vitro as measured by augmented cellular transfer of EAE to syngeneic recipients, and lymphocyte proliferation, as measured by [3]thymidine incorporation. Guinea pig (GP) MBP was approximately 60 or 1500 times more active than the GP68-84 (Y G S L P Q K S Q R S Q D E N; single-letter amino acid abbreviations) or the modified bovine (MB) 68-84 (Y G S L P Q K A Q R P Q D E N) peptides for induction of EAE, respectively. Furthermore, lymphocytes primed with either GPMBP, GP68-84, or MB68-84 crossreacted in vitro with either GPMBP, GP68-84, or MB68-84 for activation of lymphocyte transfer activity. In contrast, lymphocytes primed with either GP68-84 or MB68-84 exhibited antigen-specific proliferation in vitro exclusively in response to either GP or MB sequences, respectively. Neither GP75-84 (S Q R S Q D E N) nor GP75-86 (S Q R S Q D E N P V) induced EAE, activated lymphocytes for EAE transfer, or stimulated lymphocyte proliferation under conditions and doses tested. We conclude that (i) structurally distinct determinants, reflecting existence of functionally independent classes of antigen receptors, specify encephalitogenic and proliferative responses of primed lymphocytes and (ii) determinants for EAE induction, cellular transfer of EAE, and lymphocyte proliferation include amino acid residues in the 68-74 (Y G S L P Q K) sequence of GPMBP.

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A serum factor, cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66-71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fraction E) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractions A-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fraction F). The factor, by its retention on XM300 during ultrafiltration of fraction E, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fraction E may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66-71) of myelin basic protein with the same conformation as found in intact S24.