Enhanced transfer of experimental allergic encephalomyelitis with strain 13 guinea pig lymph node cells: requirement for culture with specific antigen and allogeneic peritoneal exudate cells

Previously, we reported that transfer of experimental allergic encephalomyelitis (EAE) with sensitized peritoneal exudate cells (PEC) in strain 13 guinea pigs is markedly enhanced if the cells are first cultured with specific antigen, myelin basic protein (BP). These cells also undergo considerable antigen-specific proliferation. In contrast, the data reported here show that lymph node cells (LNC) from sensitized animals display neither enhanced transfer nor antigen-specific proliferation after culture with BP. Enhanced transfer is obtained, however, if a second nonspecific signal is available. This second signal is provided by the presence of normal allogeneic strain 2 PEC in culture. After culture with BP and strain 2 PEC, 2.5 to 5 x 10(7) strain 13 LNC transfer disease reproducibly, in contrast with approximately 1 x 10(9) previously required for successful transfer. Addition of allogeneic or syngeneic PEC without antigen does not lead to enhanced transfer by LNC. Culture with normal syngeneic PEC plus BP oly infrequently enhances transfer by LNC. The intense mixed lymphocyte reaction (MLR) induced by addition of strain 2 PEC to strain 13 LNC precludes the use of 3H-TdR incorporation for detection of proliferation by EAE effector cells. However, inhibition of transfer with low doses of mitomycin C (2 to 5 micrograms/ml) pluse the fact that EAE effector cells are found almost exclusively in the light fraction of BSA gradients after (but not before) culture suggests that the latter are induced to proliferate in culture.     

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs.

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F₁ hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.     

Macrophage procoagulant activity as a measure of cell-mediated immunity in the mouse

Thioglycollate-induced peritoneal exudate cells (TG-PEC) developed increased procoagulant activity after incubation with lymphokine and lipopolysaccharide (LPS). Dilutions of up to 1/1000 for insoluble Con A and 1/200 for periodate-induced lymphokine supernatants were active in enhancing macrophage procoagulant activity (MPCA), which was detected after a 2-hr incubation period and steadily increased over 20 hr. MPCA could also be induced by antigen; peritoneal cells from sensitized B6AF1 mice with strong footpad reactions to ovalbumin (OVA) responded to as little as 0.1 microgram/ml OVA in the MPCA test in an antigen-specific manner. By contrast, PEC from sensitized CBA/J mice that gave poor in vivo responses to OVA only reacted with high concentrations of the antigen in vitro. Production of the lymphokine responsible for induction of MPCA required an Ly-1+2- T cell, a nylon wool-adherent cell, and an la-17-bearing adherent cell. The MPCA induced by lymphokine or LPS did not appear to be a serine esterase and was not inhibited by phospholipase C. Coagulation of human factor-deficient plasma with activated TG-PEC indicated a requirement for Factor X.     

Experimental allergic encephalomyelitis-inducing activity of synthetic polyadenylic and polyuridylic homopolymers and complexes in guinea pigs.

In addition to the capacity of polyuridylic acid (poly(U)) or complexes of polyadenylic acid (poly(A)) and Poly(U) (poly(A : U)) to serve as adjuvants for induction of experimental allergic encephalomyelitis (EAE) in guinea pigs sensitized to spinal cord or myelin basic protein, these synthetic polynucleotide homopolymers possess inherent EAE-inducing activity for this host. EAE activity of poly (A) or poly(A : U) was demonstrable following a single injection of the purine homopolymer or the complex in incomplete Freund's adjuvant (IFA). EAE-inducing activity of poly(U) was observed only in guinea pigs initially primed with this pyrimidine homopolymer in IFA.     

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.     

Immune responses to myelin basic protein in mycobacterial-induced suppression of experimental allergic encephalomyelitis

Pretreatment of guinea pigs with complete Freund's adjuvant (CFA) 21 days prior to injection with myelin basic protein (BP) in CFA resulted in marked attenuation of clinical and pathologic manifestations of experimental allergic encephalomyelitis (EAE). Delayed hypersensitivity skin tests to homologous BP were likewise depressed in protected animals. The protected guinea pigs also demonstrated diminished in vitro reactivity to BP as assessed by BP-induced proliferative response of peritoneal exudate cells (PEC) and BP-induced inhibition of macrophage migration. Broad-based suppression of immunologic reactivity did not occur in these animals, as manifested by larger skin tests to PPD, a greater proliferative response to old tuberculin (OT), by PEC and peripheral blood lymphocytes (PBL), as well as marked PPD-induced inhibition of macrophage migration. Diminution of the degree of cell-mediated reactivity to BP may be one of the mechanisms by which prior treatment with CFA suppresses subsequent development of EAE.     

Antigen-induced suppression of mitogen responses and resistance to experimental autoimmune encephalomyelitis

Random-bred Hartley and inbred Strain 2 and Strain 13 guinea pigs were inoculated for acute experimental autoimmune encephalomyelitis (EAE). Sixty-six percent (69/103) of the Hartleys developed signs of EAE while the remaining 34% (34/103) were resistant. No Strain 2 and all Strain 13 guinea pigs developed EAE. Histologic examination of nervous tissue revealed that susceptible Hartleys and Strain 13 and Strain 2 animals had lesions characteristic of EAE. Tissue from resistant Hartleys showed fewer and less severe changes. Lymphocyte-transformation assays with EAE-inducing and noninducing antigens and T-cell mitogens revealed three different sets of responses in vitro: (i) lymphocytes from all animals responded to mitogens; (ii) lymphocytes from susceptible animals responded to EAE-inducing antigens; and (iii) lymphocytes from resistant Hartleys were suppressed from responding to the mitogens solely by EAE-inducing antigens. Plasmas from all EAE-sensitized animals had equivalent anti-myelin basic proteins (MBP) antibody titers and skin tests of EAE-inoculated Hartleys were all positive for MBP sensitization. Therefore, resistance and reduced histologic changes characteristic of EAE correlated with a disease-specific antigen-induced suppression of lymphocyte responses to T-cell mitogens. This suggests that clinical resistance to EAE in Hartley guinea pigs is mediated by an immunologic suppressor mechanism.     

Immunologic activity of myelin basic protein in strain 2 and strain 13 guinea pigs.

The resistance of Strain 2 guinea pigs to experimental allergic encephalomyelitis (EAE) induced by inoculation with whole CNS tissue in complete Freund's adjuvant (CFA) has been confirmed. The resistance is even more pronounced when myelin basic protein (BP) is used in attempts to induce EAE. Strain 2 guinea pigs are also resistant to an immunization schedule (multiple injections with BP in IFA followed by a single injection of BP in CFA) known to induce significant levels of antibody in susceptible strains. The poor response of Strain 2 guinea pigs to BP is not the result of lack of specific B cells--antibody equivalent to that produced by Strain 13 animals is obtained when the inoculum contains 0.5 mg BP and 2.5 mycobacteria.     

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.     

Myelin lipophilin-induced demyelinating disease of the central nervous system

Purified lipophilin, a hydrophobic lipoprotein of myelin, induces a cell-mediated demyelinating disease of the central nervous system similar to experimental allergic encephalomyelitis (EAE) induced by the myelin basic protein (MBP). Guinea pigs challenged with lipophilin (emulsified with CFA) developed clinical and histological signs of disease indistinguishable from those developed by animals similarly challenged with MBP. Both lipophilin and MBP induced and elicited delayed-type hypersensitivity in animals challenged with respective antigens. Tryptophan, an essential component of the MBP-determinant for disease in guinea pigs, is required for the encephalitogenicity of lipophilin.     

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 suppression of mitogen responses associated with resistance to experimental autoimmune encephalomyelitis requires adherent and T cells

Resistance to experimental autoimmune encephalomyelitis (EAE) in Hartley guinea pigs has previously been reported to be associated with disease-specific antigen-induced suppression of mitogen responses in vitro. The present studies were initiated to investigate the requirement for different cell populations in this suppression. Intact and adherent-cell-depleted cultures of spleen cells from experimental and control animals were incubated with myelin basic protein (MBP), the major antigen of EAE, with the T-cell mitogen concanavalin A (Con A) alone or with Con A in the presence of MBP. In agreement with previous studies, MBP-induced suppression of the Con A response was observed only in cultures derived from resistant animals. In addition, it was observed that this suppression was abrogated by depletion of adherent cells. When cells from resistant and susceptible animals were mixed, suppression occurred only in the presence of nonadherent cells from resistant guinea pigs. Adherent cells from either resistant or susceptible animals functioned equally well. Cultures of purified E-rosette-forming cells (E+) from resistant animals (i.e., T cells) showed no suppression. Similarly, cells from these same animals which were depleted of E+ cells (i.e., non-T cells) did not demonstrate suppression in vitro. Upon reconstitution of spleen cell populations from resistant guinea pigs by mixing E+ and E- cells, suppression was restored. These experiments show that this model of suppression in vitro requires adherent cells as well as T cells and suggests that antigen-induced suppression of mitogen responses is dependent upon a cell-mediated immunologic mechanism.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

Role of macrophage-myelin basic protein interaction in the induction of experimental allergic encephalomyelitis in Lewis rats

Stimulated peritoneal exudate cells (PEC) containing activated macrophages (M phi) of Lewis (Le) rats were exposed for 1 hr in vivo or in vitro to radioiodinated soluble myelin basic protein (MBP) or MBP incorporated into magnetic microspheres (MBP-microspheres). The uptake by M phi of the dose of microsphere-bound MBP averaged 6.2%, whereas the average uptake of soluble MBP was 0.17%. Naive rats were sensitized with M phi-associated MBP or M phi-associated MBP-microspheres via the hind footpads without the aid of conventional immunologic adjuvants. Draining lymph node cells (LNC) or spleen cells from sensitized rats were cultured for 3 days with guinea pig MBP (GPMBP) alone or in combination with concanavalin A (Con A), then injected i.v. into naive recipients. Clinical signs of experimental allergic encephalomyelitis (EAE) appeared 6 days after transfer of LNC or spleen cells, and typical CNS lesions were seen in recipients sacrificed 10 to 14 days after transfer. The challenge of MO-MBP-sensitized rats with MBP-CFA resulted in severe clinical signs of EAE marked by an accelerated onset of neurologic symptoms.     

Chronic experimental allergic encephalomyelitis in guinea pigs: immunologic studies on the two major myelin proteins

Humoral and cell-mediated immunity to the two major myelin proteins, basic protein (MBP) and proteolipid protein, have been investigated during the course of chronic experimental allergic encephalomyelitis (EAE) induced in guinea pigs with whole neural tissue. A positive proliferative response to MBP was observed at 10 and 13 days postimmunization, but was not detectable at subsequent stages. Serum antibodies to MBP first appeared during the chronic stages of the disease. A proliferative response to proteolipid apoprotein was not detected during any stage of chronic EAE. Guinea pigs immunized with proteolipid alone, however, showed a proliferative response. The data suggest that MBP is one of the antigens involved in the induction of the acute episode of chronic EAE, but its role in later stages and that of proteolipid protein remain unknown.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Suppression and reversal of allergic encephalomyelitis in guinea pigs with a non-encephalitogenic analogue of the tryptophan region of the myelin basic protein.

The administration of synthetic peptide S42 leads to suppression and reversal of experimental allergic encephalomyelitis (EAE) induced in guinea pigs by myelin basic protein. Peptide S42 contains a linear sequence of 21 amino acid residues, H-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Gly-OH, made up of four repeating unit sequences of H-Phe-Ser-Trp-Gln-Lys-OH in addition to a C-terminal glycine. Injected at relatively high doses, peptide S42 is non-encephalitogenic. It induces delayed-type hypersensitivity which is not followed by EAE, and elicits delayed-type hypersensitivity responses in peptide S42, encephalitogenic trytophan peptide, or BP-challenged animals for either of the three antigens. The repeating unit sequence of peptide S42 is analogous to the encephalitogenic tryptophan region of the BP molecules . The sequence homology is responsible for cellular recognition of this antigen by the skin test assay and suggests in vivo interaction between peptide S42 and EAE-inducing cells leading to suppression and reversal of disease.     

Cellular transfer of experimental allergic encephalomyelitis in Lewis rats: Effects of different sensitization regimens on in vitro reactivity of donor spleen cells to concanavalin A

Splenocytes from Lewis rats sensitized to guinea pig spinal cord (GPSC) and complete Freund's adjuvant (CFA) or to myelin basic protein (MBP)-CFA plus pertussis vaccine were less effective than spleen cells from MBP-CFA sensitized donors in transferring EAE to syngeneic recipients following culture with concanavalin A (Con A). Moreover, splenocytes from rats sensitized to GPSC-CFA plus pertussis vaccine showed no EAE transfer activity following culture with Con A. Diminished EAE transfer activity occurred in parallel with decreased proliferative responses of primed splenocytes to Con A. These effects were due, at least in part, to the use of pertussis vaccine and to Con A activation of a suppressive adherent cell subpopulation in sensitized donor spleens. Proliferative responses and EAE transfer activity were restored upon removal of plastic-adherent cells from splenocytes of rats sensitized to MBP-CFA plus pertussis vaccine prior to Con A activation of the non adherent lymphoid cells. Deletion of plastic-adherent cells from splenocytes of donors sensitized to GPSC-CFA plus pertussis vaccine prior to activation with Con A, however, had no effect on proliferative responses or EAE transfer activity. Furthermore, EAE transfer activity of Con A-activated splenocytes from MBP-CFA-sensitized donors was lost when such cells were cultured with splenocytes from donors sensitized to GPSC-CFA plus pertussis vaccine.     

Activity of Calpain in the Guinea Pig Brain under Conditions of Experimental Allergic Encephalomyelitis

We studied the activity of calpain in the brain tissue of guinea pigs at different stages of the development of experimental allergic encephalomyelitis (EAE). Eleven days after inoculation of a mixture containing myelin main protein into the experimental animals, we observed a drop in the calpain activity (on average, by 27% with respect to the control), whereas on the 20th and 27th days the activity of the enzyme under study exceeded the norm (by 12%). The calpain/calpastatin ratio also altered at the different stages of development of EAE: the amount of calpastatin increased significantly on the 11th and 27th days, while on the 20th day the level of calpastatin was close to that typical of the control animals. Therefore, we found that the state of calpain/calpastatin system in the guinea pig brain demonstrates some correlation with the dynamics of development of EAE.