Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides.

Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.

Tolerance induction by acylated peptides: effect on encephalitogenic T cell lines.

We reported previously that acylation of an encephalitogenic peptide of myelin basic protein (MBP68-86) by attachment of palmitoyl chloride (PAL68-86) converted this peptide into a powerful tolerogen for EAE in the Lewis rat. In this study we show that T cell lines derived from a PAL68-86-protected rat proliferated poorly to MBP68-86 in vitro, even after repeated passages in this peptide and IL-2. Conversely, T cell lines derived from untreated rats that were challenged with MBP68-86 or PAL68-86 in CFA responded vigorously to MBP68-86 when propagated for many passages in this peptide but became gradually unresponsive after being propagated in the presence of PAL68-86. The modulation of the T cell lines by PAL68-86 in vitro was reflected by a significant reduction in their ability to transfer EAE to recipients. A high percentage of cells stained with an anti-Vbeta8.2 antibody, regardless of whether they were propagated in the presence of unmodified or acylated peptide. The results are consistent with the notion that tolerance induced by PAL68-86 operates by functional inactivation and provide the basis for the use of acylated peptides in the antigen-specific treatment of autoimmune diseases.

Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein.

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68-86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68-86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44-62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68-86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Native, but not genetically inactivated, pertussis toxin protects mice against experimental allergic encephalomyelitis.

Treatment of SJL mice with 400 ng Bordetella pertussis toxin (PT) either in saline or emulsified in incomplete Freund's adjuvant protected the mice against experimental autoimmune encephalomyelitis (EAE) induced 28 days later by a synthetic peptide of myelin proteolipid protein (PLP139-151) in complete Freund's adjuvant. However, treatment with a genetically inactivated pertussis toxin in which the catalytic and NAD-binding sites of the ADP-ribosyltransferase subunit were modified by site-directed mutagenesis was without effect. In vitro, lymphocyte proliferation was considerably enhanced by both the native and the inactivated toxin, at concentrations of 0.1-1 microgram/ml. However, strong inhibition of proliferation was also observed with the native toxin only, at concentrations that were two to three orders of magnitude lower than that required for the mitogenic effect (0.1-1 ng/ml). The inhibition of proliferation was detectable in the case of high-background proliferation, after stimulation with antigen (PLP139-151) or purified protein derivative of Mycobacterium tuberculosis), or with anti-CD3 monoclonal antibody, but not after stimulation with concanavalin A or phorbol esters and Ca2+ ionophore. These results suggest that the inhibitory effect of PT operates by interfering selectively with a T cell receptor-dependent signaling pathway. The biological significance of the in vitro inhibitory effect of PT was demonstrated by a considerable decrease and/or delay in the ability of lymphocytes grown with PLP139-151 and low concentrations of PT to transfer EAE to naive recipients.

Effect of anti-interferon-gamma monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains.

Immunization with myelin basic protein (MBP) in complete Freund's adjuvant failed to induce experimental allergic encephalomyelitis (EAE) in six resistant mouse strains studied: A/J, BALB/c C3H/HeJ, AKR, NZW and DBA/2. However, treatment of challenged mice with anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb) induced severe EAE in mice of all strains except AKR. Furthermore, anti-IFN-gamma mAb treatment led to increased disease incidence and severity in BALB/c mice challenged with the MBP peptide87-103, known to be encephalitogenic for the susceptible SJL strain. In three strains tested, anti-IFN-gamma mAb enhanced passively induced EAE in the A/J and C3H/HeJ but not in the BALB/c mice. All mice with clinically overt EAE had widespread histological lesions characterized by mononuclear cell infiltrates and focal demyelination. The results indicate that resistant strains are genetically capable of developing EAE, and that IFN-gamma can contribute to disease resistance.

Lewis rat T cells can reutilize, process, and present myelin basic protein to antigen-specific T cell lines.

MHC class-II-negative astrocytes prevented from intracellular antigen (Ag) processing induce myelin basic protein (MBP)-specific short-term T cell lines to proliferate. This process results from the ability of the T cells themselves to take up, process, and present Ag to each other. The Ag-presenting function of the T cells occurred in the absence of any conventional antigen-presenting cell (APC), was independent of their T cell receptor specificity, was sensitive to chloroquine, and was prevented by anti-class-II MHC antibody. Both native and HPLC-purified MBP were effective in stimulating T cell lines, and there was no obvious benefit in using either enzymatically digested or synthetic peptide preparations of the Ag. Furthermore, the Ag-presenting T cells could take up, reutilize, and re-present Ag adsorbed to the surface of histoincompatible astrocytes. Responding T cells activated by Ag-presenting T cells in the absence of other conventional APC were fully encephalitogenic upon transfer to syngeneic recipients. These results have relevance for understanding pathogenetic mechanisms in T cell-mediated autoimmunity in the central nervous system.

Rat splenocytes inhibit antigen-specific lymphocyte proliferation through a reactive nitrogen intermediate (RNI)-dependent mechanism and exhibit increased RNI production in response to IFN-gamma.

Rat splenocytes inhibited antigen-specific proliferation of primed lymph node cells in vitro. This inhibition resided in the plastic-adherent splenocyte fraction and was radioresistant, suggesting that the effect was due to macrophages. While this suppression was more evident if spleen cells were derived from immunized rats, spleen cells from normal rats were just as suppressive when added to cocultures at higher numbers. Proliferative responses were greatly enhanced in the presence of NG-monomethyl-L-arginine, a specific inhibitor of the nitric oxide synthetic pathway, and significant levels of nitrite (NO2-), a product of this pathway, were detected in culture supernatants in association with suppressed responses, supporting the notion that suppression was mediated by the L-arginine-dependent production of reactive nitrogen intermediates (RNI). When the splenocytes were physically separated from the responding lymph node cell population, high levels of NO2- were still detected but proliferative responses were no longer inhibited, suggesting that cell proximity or contact is necessary for delivery of the suppressive signal. Adherent splenocytes cultured alone produced low levels of NO2-. Addition of 1 to 50 U/ml IFN-gamma induced a dose-dependent increase in NO2- production, with the maximal level approximating that found in suppressed cocultures; TNF-alpha, IL-2, or LPS did not synergize with IFN-gamma to enhance NO2- production. These findings suggest that by activating macrophages to upregulate RNI synthesis, IFN-gamma-producing T cells may exert a negative influence over their own proliferation.

Characterization of Ca(2+)-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni.

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

Myelin-liposome protection against experimental autoimmune encephalomyelitis is associated with reduced neuroantigen-specific T-cell-mediated responses.

Lewis rats undergo a relapsing paralytic disease upon challenge with spinal cord emulsified in complete Freund's adjuvant (CFA). Treatment with two intracardiac injections of liposomes composed of whole myelin significantly reduced the severity of disease. Protection was disease-specific since treatment with myelin liposomes did not protect Lewis rats against adjuvant arthritis (AA), a CNS-unrelated T-cell-mediated autoimmune disease. Myelin-liposome-treated, spinal cord/CFA-immunized rats displayed borderline reduction of delayed-type hypersensitivity (DTH) (ear swelling) reactions to myelin and myelin basic protein (MBP), but significantly reduced in vitro lymphnode cell proliferation in response to these antigens. Responses to purified protein derivative of Mycobacterium tuberculosis (PPD) were not reduced, emphasizing the antigen-specific nature of the myelin-liposome-mediated suppression. Spleen cell proliferative responses were inconsistent and often poor. However, when cultured in the presence of NG-monomethyl-L-arginine (MMA), antigen-specific proliferation of spleen cells from both treated and control rats was greatly enhanced, indicating that reactive nitrogen intermediates contributed to the decrease in spleen cell proliferation. Purified splenic T cells from treated rats displayed a pattern of proliferation similar to that of unseparated lymphnode cells. Treatment of rats with a single injection of myelin liposomes after recovery from the first clinical episode significantly reduced the severity of the relapses.

Effect of interferon-gamma on myelin basic protein-specific T cell line proliferation in response to antigen-pulsed accessory cells.

We have shown previously that treatment of SJL/J mice with anti-interferon-gamma monoclonal antibody (mAb) exacerbated experimental allergic encephalomyelitis (EAE) only if administered at the time of encephalitogenic challenge. Here we investigate the role of interferon-gamma (IFN-gamma) and anti-IFN-gamma mAb in the early events of T cell activation in vitro. Pretreatment of murine peritoneal exudate cells (PEC) with IFN-gamma led to a significant increase in their ability to activate myelin basic protein (MBP)-specific, short-term T cell lines. When exogenous IFN-gamma was added to cocultures of T cells and MBP-pulsed PEC, the antigen-specific T cell proliferation was considerably reduced. Anti-IFN-gamma mAb added to these cultures neutralized the inhibitory effect of the exogenous IFN-gamma on T cell proliferation but had no visible effect on class II MHC expression by the antigen-pulsed PEC present in the same cultures. A reduction in T cell proliferation was also observed when the T cells were treated with IFN-gamma prior to coculture with the MBP-pulsed PEC. These results demonstrate that, on one hand, IFN-gamma enhances the ability of PEC to induce antigen-specific T cell proliferation but, on the other hand, acts on the T cells themselves by inhibiting their proliferation in response to the antigen-pulsed PEC. This may explain why treatment with anti-IFN-gamma antibody in vivo induces EAE exacerbation.

Effect of anti-interferon-gamma and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse.

SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN-gamma to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-gamma. These findings support the notion that IL-2 is required for disease induction whereas IFN-gamma plays a disease-limiting role early in the development of EAE.

Effect of anti-DNP IgG1- and IgG2a-secreting hybridomas in vivo on the development of an anti-DNP IgE antibody response in mice.

We reported previously that CBA mice pretreated with dinitrophenyl-Bordetella pertussis (DNP-BP) conjugates exhibited sharply decreased anti-DNP IgE, and increased IgG2a antibodies following immunization with DNP-ovalbumin (DNP-OA) in alum. The objective of the present experiments was to determine whether the decrease in anti-DNP IgE was attributed to a regulatory effect exerted by IgG2a antibodies. Anti-DNP monoclonal antibodies (Mab) of the IgG1 or IgG2a isotype were passively transferred to mice, 24 h before a primary immunization with DNP-OA in alum. Anti-DNP IgE production was drastically suppressed in recipients of IgG1 but not of IgG2a Mab. Similar results were obtained when the Mab were endogenously produced by intraperitoneal implantation of anti-DNP-secreting hybridomas into (BALB/cxCBA)F1 (BCF1) mice. However, neither IgG1 nor IgG2a isotypes suppressed IgE antibody production if the hybridoma implantation took place 10 days after hapten priming. These results are, to our knowledge, the first to show a clear dissociation between the effect of either passively transferred or endogenously secreted IgG1 and IgG2a antibodies in their ability to inhibit a primary anti-hapten IgE antibody response.

Treatment of spinal cord-induced experimental allergic encephalomyelitis in the Lewis rat with liposomes presenting central nervous system antigens.

Chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) in the Lewis rat, induced by the injection of spinal cord tissue in complete Freund's adjuvant (SC/CFA), was studied in vivo by treatment with liposomes containing central nervous tissue antigens, and in vitro by lymphocyte proliferation assays. Intracardiac administration of myelin basic protein (MBP) liposomes, galactocerebroside (GC) liposomes, or MBP + GC liposomes substantially reduced the clinical severity and/or delayed the onset of the initial phase of disease. Liposomes prepared from whole myelin provided even greater protection, and were effective at suppressing both the first disease episode and the relapses. These results indicate that while GC and MBP may play significant roles in the development of CR-EAE in the Lewis rat, immune responses to other antigens are probably also involved. Splenic and lymph node lymphocytes from MBP-GC liposome-treated rats, and splenic lymphocytes from cytochrome-GC (CYT-GC) liposome-treated rats, showed drastically reduced abilities to proliferate in response to MBP in culture. Spleen cells from both the MBP-GC- and CYT-GC-liposome-treated donors were able to actively suppress antigen-induced proliferation of MBP-primed lymphocytes. These findings suggest participation of both clonal anergy, and active suppressor cells in the liposome-mediated suppression of CR-EAE in the Lewis rat.

Suppression of experimental allergic encephalomyelitis by MBP-coupled lymphoid cells and by MBP-liposomes: a comparison.

In previous experiments, we showed that administration of myelin basic protein (MBP) inserted into phosphatidyl-serine liposomes, to susceptible animals suppressed the clinical manifestations of both acute and chronic-relapsing EAE. In this report we compare the effectiveness of treatment with MBP-liposomes and with MBP-coupled syngeneic spleen cells in EAE protection. Lewis rats treated with 150 micrograms MBP-liposomes or with 160 micrograms (35 x 10(6] MBP-coupled spleen cells, given 7 days before and 7 days after encephalitogenic challenge were equally protected against clinical EAE, when compared to untreated controls. In addition to clinical protection, in vitro proliferative responses of lymphocytes from treated rats were significantly reduced, but delayed hypersensitivity (DTH) reactions remained unaffected. Proliferation of lymphocytes from MBP-sensitized donors was inhibited by the addition of spleen cells but not of lymph node cells from treated donors. The inhibitory effect was observed with spleen cells regardless of whether the donors were treated or not, was antigen nonspecific, and localized in a radio-resistant, adherent cell population. Adoptive transfers of spleen cells from treated donors, after a 48-hr in vitro incubation with concanavalin A, showed that the cells from donors treated with MBP-coupled spleen cells, but not with MBP-liposomes, suppressed the disease in recipients, following challenge with MBP-complete Freund's adjuvant (CFA). These results suggest that two distinct mechanisms operate in the protection by MBP-coupled cells and MBP-liposomes, respectively.

Role of interferon-gamma in the modulation of the IgE response by 2,4-dinitrophenyl-Bordetella pertussis vaccine in the mouse.

Immunization of mice with 2,4-dinitrophenyl-Bordetella pertussis (DNP-BP) failed to induce anti-DNP IgE responses. Administration of DNP-BP induced, however, the formation of anti-DNP IgE B memory cells, as demonstrated by adoptive transfer. Furthermore, mice pretreated with DNP-BP and primed with 2 micrograms DNP-ovalbumin (OA) in alum 2 weeks later produced high day-7 anti-DNP IgE levels. These subsided to near undetectable levels by day 12-14. The transient expression of serum IgE levels was accompanied by normal levels of anti-DNP IgG. The anti-OA response induced as a result of priming with DNP-OA in alum was not affected by pretreatment with DNP-BP. IgG subclass analysis revealed that mice pretreated with DNP-BP had elevated levels of IgG2a and reduced levels of IgG1 as compared to control (TNP-keyhole limpet hemocyanin-pretreated) mice. Treatment of mice with an anti-interferon-gamma monoclonal antibody, shortly after immunization with DNP-BP, not only reduced anti-DNP IgG2a levels, but prevented the sharp anti-DNP IgE decline that occurred after priming with DNP-OA in alum. These results suggest that DNP-BP-induced interferon-gamma production modulates Ig isotype expression in vivo in an anti-gen-specific manner.

Chronic-relapsing experimental allergic encephalomyelitis in strain-13 guinea pigs: cell-mediated immunity and IgG isoelectric focusing in myelin basic protein-liposome-treated and untreated animals.

Juvenile strain-13 guinea pigs challenged with whole central nervous system (CNS) tissue in complete Freund's adjuvant (CFA) developed chronic-relapsing (CR) experimental allergic encephalomyelitis (EAE). The animals that recovered from the first clinical episode were divided into three groups. One group was left untreated, one group was treated with three intracardiac injections of 100 micrograms glutaraldehyde-fixed myelin basic protein (MBP)-liposomes (MBP-L-GA) given once a week, and one group was treated with cytochrome c-liposomes (CYC-L-GA). The animals treated with MBP-liposomes were very well protected against further relapses. In vitro proliferative responses of peripheral blood lymphocytes (PBL) were performed repeatedly on most animals. The lymphocytes exhibited excellent proliferative responses to MBP, proteolipid apoprotein (PLP) and whole myelin, as well as to purified protein derivative (PPD) and concanavalin-A (ConA). High proliferative responses were recorded over the entire period of observation which lasted 12-22 months, each time the animals were tested in remission or in full relapse. However, a sharp decrease in proliferative responses was observed in most animals when the assay was performed 24-48 h before to 24 h after entering a relapse. The results demonstrate the presence of long-term and sustained cell-mediated responses to two distinct neuroantigens, and show fluctuations of both neuroantigen-specific and nonspecific responses concordant with a well-defined phase of the disease. Isoelectric focusing and immunofixation was performed on sera and cerebrospinal fluids obtained at the time of sacrifice. The pattern showed clear oligoclonal IgG bands (OB) in the samples obtained from untreated, CYC-L-GA-treated as well as in the MBP-L-GA-treated animals.

Effect of treatment with glutaraldehyde-fixed myelin basic protein-liposomes on active induction and passive transfer of experimental allergic encephalomyelitis in Lewis rats.

Guinea pig myelin basic protein (MBP)-liposomes were prepared and fixed with 0.2% glutaraldehyde (GA). Lewis rats were treated with glutaraldehyde-fixed MBP-liposomes (MBP-L-GA) or with cytochrome-c-liposomes (CYC-L-GA), 7 days before and 7 days after challenge with MBP in CFA. Rats treated with MBP-L-GA, but not with CYC-L-GA, were very well protected against the clinical manifestations of EAE. The protection was better than that obtained after treatment with conventional MBP-liposomes (without glutaraldehyde). Furthermore, when grown in vitro for 72 hr in the presence of MBP, lymphocytes from rats treated with MBP-L-GA and challenged with MBP in CFA exhibited a marked decrease in their ability to transfer EAE to normal syngeneic recipients.

Antigen leakage from immunosorbents. Implications for the detection of site-directed auto-anti-idiotypic antibodies.

The detection of site-directed anti-idiotypic antibodies is usually based on their ability to inhibit the binding of antigen to idiotype, in either solid- or fluid-phase radioimmunoassays. Passage of serum over antigen-coupled immunosorbents for the purpose of removing the idiotypes from complexes with putative anti-idiotypic antibodies resulted in the release of significant amounts of antigen into the effluents. Normal sera or even isotonic buffers were similarly contaminated with antigen. The amount of antigen released ranged between 200-400 ng/ml, well in excess of the minimal amount required in the inhibition assay. Antigen was detected in effluents passed over a number of antigen coupled-matrices and even in affinity-purified antibody preparations obtained by elution from immunosorbents coupled with dinitrophenyl (DNP)-protein conjugates. Attempts to stabilize the antigen-coupled matrices with glutaraldehyde resulted in a perceptible but insufficient decrease in the amount of antigen released. In the case of anti-hapten antibodies, antigen interference was circumvented by utilizing monovalent haptens such as DNP-lysine coupled to the immunosorbent either directly or through a spacer arm. In the case of protein antigens, the leakage was almost completely prevented by preparing glutaraldehyde-polymerized immunosorbents directly from solution.

The role of myelin lipids in experimental allergic encephalomyelitis. III. Transfer of suppression from guinea pigs recovering from EAE, induced by myelin basic protein--galactocerebroside complexes.

Juvenile strain 13 guinea pigs were immunized with myelin basic protein (MBP) combined with galactocerebrosides (MBP + GC) or with total myelin lipids without GC [MBP + (TL-GC)] in CFA. Control animals received dinitrophenylated-ovalbumin (DNP-OA) in CFA, CFA or IFA alone. The animals injected with MBP + GC showed a higher rate of recovery from the first EAE episode (83%) than those treated with MBP + (TL-GC) (50%). With the exception of the group treated with IFA alone, all animals were refractory to EAE following rechallenge with MBP in CFA 90 days after the first exposure. The in vitro proliferative response to MBP, of peripheral blood lymphocytes (PBLs) derived from guinea pigs freshly sensitized to MBP in CFA, was drastically suppressed in the presence of PBLs from animals injected with MBP + GC. Upon transfer to normal syngeneic recipients, spleen cells from MBP + GC-treated animals completely suppressed the clinical and histological manifestations of EAE following recipient challenge with MBP in CFA. Cell-free supernatants from PBLs and spleen cells of strain 13 guinea pigs treated with MBP + GC inhibited lymphocyte proliferation to MBP, of allogeneic responder cells, and spleen cell supernatants completely suppressed the induction of EAE upon transfer to allogeneic recipients. Suppression could not be transferred with cells from other treated groups. These results suggest that animals immunized with MBP + galactocerebrosides in CFA develop suppressor cells that may be in part responsible for the recovery from the first EAE episode and for protection against rechallenge with MBP in CFA. Their cell-free supernatants act in an MHC-nonrestricted fashion. These results do not rule out an additional protective mechanism since all animals exposed to CFA were refractory to rechallenge despite lack of demonstrable suppressor cell activity.