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.     

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.     

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.     

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.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

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.     

Strain differences in guinea pigs' bronchial sensitivity to acetylcholine

The bronchial sensitivity to acetylcholine (ACh) of guinea pigs of various strains was investigated to clarify strain differences. Inbred Strain 2, Strain 13 and JY-1 and non-inbred Hartley strain (two colonies) were used in this experiment. (1) Guinea pigs were exposed to 0.08% ACh aerosol and the time needed to produce falling down (TNPFD) was determined. Mean +/- standard error of TNPFD (n = 14 per group) of animals was 182 +/- 28 sec, 148 +/- 22 sec, 210 +/- 30 sec, 342 +/- 24 sec and 406 +/- 36 sec in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. There was a significant difference in TNPFD between inbred strains and non-inbred strains (P less than 0.05 or P less than 0.01), indicating that inbred strains had higher sensitivity. (2) Guinea pigs were exposed to 20-5000 micrograms/ml ACh for 2 min. The mean dose threshold as determined by transcutaneous oxygen pressure was 524 micrograms/ml, 424 micrograms/ml, 614 micrograms/ml, 1317 micrograms/ml and 1651 micrograms/ml (n = 14 per group) in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. Inbred strains showed lower dose thresholds than non-inbred strains. (3) Isolated trachea-lungs of 5 guinea pigs were perfused with 10(-9)-10(-5) g/ml ACh to determine strain differences. Dose response curves of animals of inbred strains shifted to the left (lower concentrations), unlike those of non-inbred strains, suggesting that inbred strains had higher sensitivity to ACh than non-inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Susceptibility and resistance to experimental autoimmune encephalomyelitis and neuritis in the guinea pig correlate with the induction of procoagulant and anticoagulant activities

Activation of macrophage procoagulant activity (MPCA) is involved in the manifestation of EAE and EAN in susceptible guinea pigs and provides a mechanism for the deposition of fibrin, which is a feature of histologic lesions of EAE. Peritoneal exudate cells (PEC) from susceptible (strain 13) guinea pigs immunized with either central or peripheral nervous tissue antigens produce procoagulant activity when incubated with the immunogen in vitro. The production of the procoagulant is quantitative and antigen-specific and is maximal at the time of clinical signs of the disease. After recovery, the production of procoagulant activity decreased. The MPCA test was able to discriminate the biochemical differences existing between chicken and mammalian peripheral nerve proteins, thus providing a quantitative and sensitive indicator of cell-mediated immunity in EAE and EAN. The autoimmune response to brain and nerve antigens in nonsusceptible (strain 2) guinea pigs was coincident with the antigen-specific production of a cell-bound anticoagulant activity by stimulated mononuclear cells. The production of anticoagulant activity followed the same sequence of time changes after immunization as that of the MPCA in susceptible guinea pigs, and high immunizing doses of nerve antigens induced high levels of anticoagulant activity. The same cells produced high levels of procoagulant when incubated with tuberculin or lipopolysaccharide. The recalcification time of normal plasma was prolonged by the anticoagulant, and the decreased clotting time of plasma induced by the procoagulant activity obtained by incubating sensitized strain 13 PEC with myelin basic protein was suppressed by the anticoagulant produced by culturing sensitized strain 2 PEC with myelin basic protein. Preliminary evidence indicates that the anticoagulant has properties similar to antithrombin III. The anticoagulant could play a role in the control of effector cell function, and therefore in recovery from clinical features of EAE and EAN in susceptible guinea pigs.     

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.     

In vitro analysis of immune responses of inbred guinea pigs infected in vivo with Leishmania enriettii and an investigation of active immunization of susceptible animals

Adherent skin cell monolayers have been prepared from the infected area of inbred guinea pigs inoculated with Leishmania enrietti. Cells from those animals most susceptible to disease (2/N) are less able to promote proliferation and the production of macrophage migration inhibition factor (MIF) from leishmania-immune lymphocytes than are infected cells taken from the more resistant 13/N or (2/N × 13/N)F₁ animals. Exposure of immune lymphocytes of all strains to parasite antigen in the relative absence of autologous antigen-presenting cells induced the development of a suppressor pool capable of inhibiting lymphocyte proliferation in response to immunogenic signals. Decreased lymphocyte proliferation during the course of disease may be caused by the endogenous release of products of arachidonic acid metabolism from host monocytes, though along with the decline in DTH reactivity in infected animals, and apparently in concert with the healing of the primary lesion, there occurs an increase in titer of antileishmania antibody in infected animals. Preliminary attempts to confer protection of naive animals by preimmunization with lymphocytes from previously infected, susceptible, or resistant guinea pigs suggested a role for interacting “helper” and suppressor lymphocyte pools in the immunoprotection from leishmania infection.     

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.     

A comparison of autoimmunity and experimental allergic thyroiditis in strain 2 and Hartley strain guinea pigs

The response of Strain 2 guinea pigs to immunization with thyroid extract in complete adjuvant was compared with the response of the Hartley strain. The Strain 2 did not develop thyroiditis as frequently or in as great a degree as the Hartley strain. This difference in degree of thyroiditis between these two strains occurred consistently even when the animals were immunized with a wide range of doses of thyroid extract and of mycobacteria in adjuvant, and was independent of the strain origin of the antigen used for immunization. Although the Strain 2 made less thyroiditis than the Hartley strain, it made as much agglutinating antibody, and often as much delayed sensitivity as the Hartley strain. The genetic controls regulating autoimminity to the thyroid and production of thyroiditis appear to differ from those regulating production of experimental allergic encephalomyelitis.     

Suppression of experimental autoimmune thyroiditis in guinea pigs by pretreatment with thyroglobulin-coupled spleen cells

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG) coupled to syngeneic spleen cells (GPTG-SC) suppressed the development of experimental autoimmune thyroiditis (EAT) induced by immunization with GPTG in complete Freund's adjuvant (CFA). Antibody titers to GPTG were only minimally suppressed in GPTG-SC pretreated animals. GPTG-SC also suppressed the sensitization of periotneal exudate T lymphocytes which proliferate in vitro in the presence of GPTG.     

Unusual pattern of surface marker expression on peripheral lymphocytes from aged humans suggestive of a population of less differentiated cells

SJL/J, PL/J, and (SJL/J x PL/J)F1 mice were immunized with bovine, guinea pig, mouse, or rat myelin basic proteins (MBP) in adjuvant containing Mycobacterium tuberculosis H37Ra. Twenty-four and 72 hr later, Bordetella pertussis vaccine was given i.v. All MBP tested induced experimental allergic encephalomyelitis (EAE) in SJL/J and F1 mice; however, bovine MBP was inactive in PL/J mice. Each strain was immunized in a similar manner with peptic peptides, residues 1-37, 43-88, and 89-169 of guinea pig MBP. In contrast to the SJL/J strain, which has been shown to recognize a major encephalitogenic determinant in peptide 89-169, PL/J and F1 mice responded primarily to an encephalitogenic determinant within peptide 1-37. Analysis of antibody levels showed that SJL/J mice made no antibody to peptide 1-37, although anti-peptide 89-169 antibodies were consistently found. Conversely, PL/J mice responded well to peptide 1-37, but only an occasional animal made a significant response to peptide 89-169. (SJL/J x PL/J)F1 mice were more susceptible to EAE than either parental strain, as shown by the percentage of animals showing neurologic signs and by clinical severity.     

Immune response gene control of antibody specificity

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F₁ (2 × 13) guinea pigs, immunized with the αDnp-Lys₉, produce both T cells and antibody which are equally discriminatory for αDnp-Lys₉. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with αDnp-Lys₉ do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F₁ (2 × 13) environment, PLL Ir gene-negative B cells are provided with F₁ (2 × 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.     

Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects

Experimental allergic encephalomyelitis (EAE)-susceptible Lew and EAE-resistant Brown Norway (BN) rats and the corresponding MHC congenic strains were examined for their ability to develop clinical and histologic EAE. The ability of T cells from these animals to proliferate in vitro in response to whole guinea pig (GP) myelin basic protein (MBP), rat MBP, and to the major encephalitogenic peptide of GP MBP 66-88 (GP 68-88) was also assessed. We found that Lewis (Lew) was highly susceptible and showed good T cell responses to GP, MBP, rat MBP, and GP 68-88. Lew.1N (BN MHC on Lew background) and BN were not susceptible and T cells from these strains showed significant responses to GP MBP, but not to rat MBP or GP 68-88. Although BN.B1 (Lew MHC on BN background) was not susceptible to actively induced EAE, MBP-specific Lew T cells could transfer severe disease to BN.B1. BN.B1 T cells showed responses to GP-MBP, rat MBP, and GP 68-88 and, when transferred to naive BN.B1 or Lew, induced only mild clinical EAE in both strains. Increasing the number of T cells from BN.B1 had no effect on the severity of clinical symptoms in either recipient, suggesting some deficiency in the T cell repertoire that is necessary for induction of severe EAE. These results suggest that 1) the T cell response to rat MBP and GP68-88 (but not to sites other than 68-88 in GP MBP) is necessary for susceptibility to EAE; 2) the ability to respond to both rat MBP and GP 68-88 is determined by the MHC gene products on APC; and 3) given a permissive MHC, the T cell response that results in EAE is influenced by non-MHC genes.     

Induction of allergic reactions in guinea pigs with purified house dust mite allergens

To establish a guinea pig model for house dust mite allergy with purified mite allergens, we studied the immune response to two major mite allergens, native Der f 1 (nDer f 1) and recombinant Der f 2 (rDer f 2) and crude mite extract in Hartley guinea pigs. Animals were immunized with either mite extract, nDer f 1 or rDer f 2, four times at 2- to 3-week intervals. Then the guinea pigs were examined as to the status of sensitization to the sensitizing antigen. Intradermal injection of mite antigens to mite extract-, nDer f 1-, and rDer f 2-sensitized animals induced both immediate and late-phase cutaneous reactions. Allergic airway disease was also provoked by the intranasal instillation of rDer f 2 or mite extract. Anti-nDer f 1 and -rDer f 2 IgE as well as anti-mite extract IgE were produced in the sensitized guinea pigs and IgE titer for three mite antigens were comparable. We concluded that immunization of Hartley guinea pigs with nDer f 1 and rDer f 2 achieved sensitization to mite allergens, which was comparable to that obtained by the immunization with mite extract. A mite-allergic model suitable for immunological and pharmacological studies was established from rDer f 2-sensitized guinea pigs.     

Immune responses of inbred guinea pigs against random terpolymers containing L glutamic acid and L alanine.

The immune responses of the inbred guinea pig strains 2 and 13 have been determined against random terpolymers of L glutamic acid and L alanine and a third amino acid. Strain 2 guinea pigs responded against GAT10, GAT20(LLD), GAT10(NO2)15, GAT4, and GAL10. However, strain 13 guinea pigs responded only against GAT10. The explanation offered is that strain 2 guinea pigs, which have the Ir-GA gene, recognize the polymers via random GA determinants present in sufficient concentration in all of the above polymers. However, strain 13 guinea pigs recognize the GAT10 via the Ir-GT gene, and reduction in the concentration of tyrosyl residues below 10 mole % by various procedures alters the concentration of available random GT determinants necessary for interaction with the gene product of the Ir-GT gene.