Antibody responses in chronic relapsing experimental allergic encephalomyelitis: correlation of serum demyelinating activity with antibody titre to the myelin/oligodendrocyte glycoprotein (MOG)

Antibody responses to the myelin/oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) were determined in the sera of Hartley guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE) using an enzyme-linked immunoassay. The sera were also tested for in vivo demyelinating activity by infusion into the subarachnoid space of normal rats. In contrast to the MBP titres, the anti-MOG antibody titres showed good correlation with the in vivo demyelinating activity of the sera (r = 0.91, P less than 0.001). This result suggests that antibodies directed against MOG may be involved in the pathogenesis of demyelination in CREAE.     

In vivo demyelinating activity of sera from animals with chronic experimental allergic encephalomyelitis. Antibody nature of the demyelinating factor and the role of complement

Sera from guinea pigs and rats with chronic experimental allergic encephalomyelitis were injected into the cerebrospinal fluid (CSF) of normal recipient rats. Guinea pig sera induced demyelination in the central and (or) peripheral nervous system, whereas injection of rat sera resulted in demyelination in the peripheral nervous system only. Control sera did not induce demyelination. Demyelinating activity in guinea pig sera was confined to the IgG-fraction; in rat sera the IgG- as well as the IgM-fraction were able to induce demyelination. The demyelinating activity was abolished when the sera were absorbed with with sensitising antigen (guinea pig spinal cord tissue) or when immunoglobulins were removed from the sera. When chronic EAE sera from rats were injected into the CSF of rats, complement was not required for the induction of demyeLination. The presence of complement, however, augmented the demyelinating activity. Decomplemented chronic EAE sera from guinea pigs failed to induce demyelination after injection into the CSF of rats. Injection of control and non-demyelinating or demyelinating EAE sera into the subarachnoid space of normal recipient rats induced a weak inflammatory response with increased numbers of large mononuclear cells in the meninges. It is discussed that in vivo a complex interaction of antibodies, complement and effector cells is responsible for induction of demyelination.     

Acute canine idiopathic polyneuropathy (ACIP) serum demyelinates peripheral nerve in vivo

We examined the in vivo demyelinating activity of serum from dogs with acute canine idiopathic polyneuropathy (ACIP), a Guillain-Barré syndrome (GBS)-like illness. Sera from 6 ACIP cases demyelinated rat sciatic nerves more intensely than 11 control sera. Serum activity increased after guinea pig serum (gps) was added, although gps alone had little effect. ACIP sera did not bind more to whole nerve cross sections or Schwann cells in vitro than control sera, and did not contain elevated antigalactocerebroside titers. We do not as yet know the pathogenic importance of the demyelinating factor in ACIP and control dog serum, or understand its relationship to the demyelinating constituent in serum from humans with GBS.     

In vivo effect of sera from animals with chronic relapsing experimental allergic encephalomyelitis on central and peripheral myelin

Summary Sera from guinea pigs with acute or chronic relapsing experimental allergic encephalomyelitis (EAE) were injected into the lumbosacral subarachnoid space of normal recipient rats. Seventeen of 37 sera induced demyelination in the CNS, and 27 of 37 sera caused demyelinated peripheral nerve fibers in the roots. The highest incidence of demyelinating activity of EAE sera was noted in those from donor animals sampled during the early chronic stage of the disease [40–100 days post sensitization (dps)]. Only few sera from animals sampled during the acute and subacute stage (10–40 dps) were able to induce demyelination. Sera from animals sampled between 100 and 200 dps showed a lower incidence of demyelinating activity as compared to those from the early chronic phase of the disease. There was no clear-cut correlation between the serum-demyelinating activity and the severity of the demyelinating disease in the donor animals. The patterns of demyelination in the central as well as peripheral nervous system of recipient animals were characterized by vesicular disruption of myelin or myelin stripping. Myelin degradation was performed mainly by macrophages. In the CNS some astrocytes also contained debris. Astrocytes increased in size, and mitosis of astrocytes was observed. Oligodendrocytes appeared to be unaffected. No demyelination was found when the sera from animals sensitized with CFA alone or with guinea pig liver tissue were injeted into the subarachnoid space of normal recipient rats.Two possible mechanisms of demyelination are diseussed: Antibody-mediated complement-dependent and antibody-dependent cell-mediated demyelination.Supported in part by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria, Project No. S-25/07     

Chronic relapsing experimental allergic encephalomyelitis in SJL mice following the adoptive transfer of an epitope-specific T cell line

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in SJL mice following the adoptive transfer of a T cell line derived from mice immunized with a synthetic peptide corresponding to residues 89-100 of the guinea pig myelin basic protein (MBP) molecule. This cell line proliferated to both the peptide and MBP and induced CREAE characterized by a series of relapses with eventual stabilization. Central nervous system (CNS) inflammation and demyelination were prominent neuropathologic features of both the acute and relapsing phase of the disease. The chronic phase was characterized by CNS lesions containing chronically demyelinated fibers with remyelination and some fiber drop-out, but little inflammation. The induction of CREAE in SJL mice by a cell line specific for residues 89-100 demonstrates that T cell recognition of this epitope is required for successful disease induction in this strain of mouse.     

Immune response to isolated oligodendrocytes

Oligodendrocytes were isolated from bovine white matter and were injected with complete Freund's adjuvant (CFA) into experimental animals. Indirect immunofluorescence studies using fluoresceinated goat anti-rabbit or anti-guinea pig immunoglobulin (GARIg; GAGPIg) showed that rabbit and guinea pig anti-oligodendrocyte (RAO, GPAO) sera reacted specifically with the surface of isolated oligodendrocytes in suspension, as well as with oligodendroglia in bovine and human brain sections, and in mouse cerebellum cultures. This activity of RAO was blocked by non-fluoresceinated GARIg and by GPAO, and absorbed by oligodendrocyte preparation (OP) or whole white matter, but not by purified myelin, neuroblastoma or non-brain tissue. Low levels of anti-basic protein antibodies were found in many RAO (but not GPAO) sera by radioimmunoassay, and a few showed significant anti-galactocerebroside antibody by agglutination and radioimmunoprecipation techniques. Guinea pigs sensitized with isolated oligodendrocytes in CFA showed cell-mediated immunity (CMI) to OP as manifested by delayed type skin test and induced in vitro lymphocyte transformation. CMI to purified myelin basic protein was not detected. The demonstration of humoral and CMI to the cell responsible for the production of CNS myelin may be related to some aspects of the immunopathogenesis of demyelinating disorders.     

Peripheral nervous system lesions in experimental allergic encephalomyelitis

Summary The distribution of T cells and Ia-antigen in peripheral nervous system (PNS) lesions of experimental allergic encephalomyelitis was studied by light- and electron-microscopic immunocytochemical techniques. Sprague Dawley rats, sensitized with guinea pig spinal cord tissue, developed a biphasic disease with acute inflammatory and chronic inflammatory demyelinating lesions in the PNS. In both the acute non-demyelinating and the chronic demyelinating disease inflammatory infiltrates were composed of T cells and Ia-positive monocytes/macrophages. Dependent upon the stage of the disease a variable percentage of T-lymphocytes carried the Ox 8 antigen (suppressor/cytotoxic cells). In demyelinating lesions no evidence for an interaction of T cells with myelin or Schwann cells was observed, thus arguing against a direct T-cell cytotoxicity in demyelination. The whole sequence of myelin destruction and digestion was performed by W3/13^–, Ia⁺ mononuclear cells with ultrastructural features of monocytes/ macrophages. In contrast to the acute inflammatory stage of the disease, high titers of anti-myelin antibodies were present in sera of affected animals sampled during the chronic inflammatory demyelinating stage. The sera from the latter animals also showed pronounced in vivo demyelinating activity when transferred into the cerebrospinal fluid (CSF) of normal recipient rats. It is thus suggested that demyelination in this model is induced by a co-operation of cell-mediated and humoral immune mechanisms.We did not find evidence for Ia-antigen expression on local elements of the PNS (Schwann cells, axons, or endothelial cells).Supported in part by the Austrian Science Research Fund, proj. no. P5354     

Chronic experimental autoimmune encephalomyelitis. Circulating autoantibodies bind predominantly determinants expressed by complexes of basic protein and lipids of myelin

Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was induced by immunising juvenile strain 13 guinea pigs with homologous spinal cord tissue in adjuvant. Thirteen animals were killed in the early chronic (5-12 weeks post immunisation) and 8 in the late chronic phase (after 15 weeks pi). Plasma titres of antibodies to an isolated myelin preparation were determined by enzyme linked immunoassay. Elevated titres of these antibodies were detected between 5-26 weeks pi, varied by 10-fold between different individuals, and had no direct relationship to clinical status or time pi. Of 20 CREAE plasma with anti-myelin immunoglobulins (Igs), only 3 contained substantial amounts of antibodies to myelin lipid and these were all from animals in the late chronic phase. By contrast 15/20 of the samples contained antibodies which appeared to require lipid-protein interactions for optimal binding to antigens in isolated myelin. There was a close correlation between plasma titres of antibodies to isolated myelin and to purified myelin basic protein (MBP). Even in samples with a moderately high lipid requirement for binding to isolated myelin, purified MBP could inhibit at least 50% of the binding. These observations suggest that MBP-lipid complexes are dominant immunogens in CREAE. Gross inflammation and myelin loss in spinal cords from these CREAE guinea pigs were determined by light microscopy. Substantial inflammation was apparent in some animals between 7 and 26 weeks pi. The most severe myelin loss was observed in late chronic phase animals having plasma anti-myelin Igs with a variety of specificities. The data suggest that circulating antibodies to myelin lipids or MBP-lipid complexes could contribute to demyelination in CREAE but their titres do not correlate with the extent of this process.     

Brain-derived gangliosides suppress the chronic relapsing-remitting experimental autoimmune encephalomyelitis in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Chronic relapsing-remitting experimental autoimmune encephalomyelitis (CREAE) induced with myelin oligodendrocyte glycoprotein peptides 35-55 (MOG(35-55)) in NOD mice was successfully treated with brain-derived gangliosides (GA). The GA treatment suppressed the development and severity of CREAE, both clinically and histologically. Spleen cells from the GA-treated mice displayed markedly inhibited levels of MOG(35-55) specific proliferation and interferon-gamma production. Delayed-type hypersensitivity reactions to MOG(35-55) were suppressed by the GA treatment. GA modulate various T cell effector functions in CREAE and may be an effective therapeutic agent for autoimmune demyelinating diseases such as multiple sclerosis.     

Immunologic studies of factor VIII coagulant activity (VIII:C). 2. Factor VIII in selected vertebrates

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.     

Mechanism of demyelination in the guinea pig

Experimental allergic encephalomyelitis (EAE), accompanied by demyelinating central nervous system (CNS) lesions, can be induced in guinea pigs sensitized with whole guinea pig CNS tissue, but not in animals sensitized with purified myelin basic protein (BP). This type of chronic demyelinating EAE is presumably a result of a combination of a cell-mediated immune response to the encephalitogenic BP and a separate response to other nonencephalitogenic CNS antigens. We report here that demyelinating EAE can be induced when separate sensitizations are used to induce a cell-mediated response to BP and a second immune response to nonencephalitogenic CNS antigens. Animals sensitized in separate sites with guinea pig BP and whole chicken brain develop CNS demyelinating lesions. Animals sensitized only to BP or chicken brain do not develop demyelination. The antigen(s) responsible for demyelination are found in the myelin fraction of chicken brain.     

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin antigens: IV. Suppression of chronic relapsing disease in the Lewis rat and strain 13 guinea pig.

Oral administration of proteins is a long-recognized method of inducing antigen-specific peripheral immune tolerance. We previously showed that oral administration of myelin basic protein suppresses monophasic experimental autoimmune encephalomyelitis in the Lewis rat when it is given in association with immunization and prior to disease onset. As a potential therapy for human autoimmune disease, it is crucial to determine whether oral tolerance can ameliorate an ongoing immune response. We therefore asked whether oral administration of myelin antigens, after sensitization and disease expression has occurred, could affect immunological, clinical, or pathological features of experimental autoimmune encephalomyelitis. Chronic relapsing experimental autoimmune encephalomyelitis was induced in the Lewis rat and strain 13 guinea pig by immunization with whole guinea pig cord homogenate, complete Freund's adjuvant, and Mycobacterium tuberculosis. Following recovery from the first attack, animals were orally given bovine myelin, guinea pig myelin, or guinea pig myelin basic protein three times per week for up to 3 months. Animals receiving myelin products orally had decreased severity and frequency of clinical relapses, decreased delayed-type hypersensitivity responses to myelin antigens, diminished inflammation in the central nervous system (CNS), and decreased areas of CNS demyelination. In the rat, guinea pig myelin basic protein was as effective as guinea pig myelin in ameliorating the disease and also resulted in decreased serum anti-myelin basic protein antibody levels. No exacerbation of disease or worsening of pathological findings occurred in the animals given myelin products. These results demonstrate that oral administration of myelin antigens can suppress chronic relapsing experimental autoimmune encephalomyelitis and have direct relevance to therapy of human demyelinating disorders such as multiple sclerosis.     

Serum antibodies against glycosphingolipids in chronic relapsing experimental allergic encephalomyelitis. Demonstration by ELISA and relation to serum in vivo demyelinating activity

Sera from guinea pigs with spinal cord-induced chronic relapsing experimental allergic encephalomyelitis (crEAE) were tested for IgG antibodies against glycosphingolipids (GSL; galactocerebroside, ganglioside GM1, sulfatide) by an enzyme-linked immunosorbent assay and for in vivo demyelinating activity by infusion into the lumbosacral subarachnoid space of normal rats. In chronic stage-crEAE sera (40-200 days after sensitization) a high incidence (21/26) and high titers (up to 1:2560) of antibodies against one or more GSL coincided with a high incidence (22/26) of in vivo demyelinating activity. These results suggest an involvement of antibodies against various GSL in the process of demyelination.     

Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine

Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 × 10⁻⁷ M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS.     

Complement-dependent lysis of cultured rat myotubes by myasthenic immunoglobulins

We used cultured myotubes to demonstrate complement-dependent lysis of muscle membranes by serum from patients with myasthenia gravis. Lysis was monitored by light microscopy and release of incorporated [86Rb]. In the presence of guinea pig complement (GPC), 18 of 37 heat-inactivated myasthenic sera (49%), but none of 16 controls, caused morphologically detectable myotube lysis. Ten of 19 myasthenic sera (53%) increased [86Rb]-release compared with 10 controls. Immunoglobulin fractions retained the complement-dependent lytic activity. Inactivation of GPC prevented the lysis. [86Rb]-release appeared to correlate with clinical severity. The complement-dependent lysis resulted in a decrease in the number of acetylcholine receptors (AChRs) in myotubes, and AChR-immunoglobulin complexes were found in the medium of lysed cultures. The data suggest that cultured myotubes can be used to document complement-dependent antibody reactions in the pathogenesis of myasthenia.     

Anti-myelin-associated glycoprotein antibody in sera from patients with demyelinating diseases

An enzyme-linked immunosorbent assay (ELISA) was developed for quantitating anti-myelin-associated glycoprotein (MAG) IgM antibody in human sera. Absorbance values of anti-MAG antibody were higher than 0.2 at 1:80 of serum dilution in sera from some patients with demyelinating diseases of the central or peripheral nervous systems including multiple sclerosis, subacute sclerosing panencephalitis, Guillain-Barré syndrome, chronic relapsing polyradiculoneuritis and carcinomatous polyneuropathy and also some patients with autoimmune diseases such as collagen diseases and myasthenia gravis. However, absorbance values of anti-MAG antibody in sera from control individuals and patients with some other neurological diseases were less than 0.2 and considered as negative. Because of the reported existence of a cross antigenicity between MAG and lymphocyte, and especially natural killer cells, the possibility of the functional importance of anti-MAG antibody on cellular immunity is discussed with particular reference to the demyelinating diseases.     

Activation of guinea pig platelets by a monoclonal antibody

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [³h]serotonin release, and platelet protein phosphorylation. GT212 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of ³²P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indometacin. The F(ab′)₂ fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.     

Complement-dependent toxicity of serum from myasthenic patients to muscle cells in culture

The toxicity of myasthenic sera to rat myotubes in monolayer culture was examined by measuring the release of [Me-3H]carnitine from pre-loaded cells. In the presence of guinea pig complement, heat-inactivated serum samples from 9 out of 13 myasthenic patients showed clear myotoxicity, in contrast to 0 out of 11 normal controls and 0 out of 6 polymyositis patients. Neither heat-inactivated sera alone nor guinea pig complement sera alone showed myotoxicity. Removal of anti-acetylcholine receptor (anti-AChR) antibodies from a myasthenic serum sample by affinity absorption led to loss of myotoxicity. Myotoxicity of myasthenic sera could, in most cases, be confirmed by light microscopy. These results support the idea that complement-mediated cell damage, initiated by anti-AChR antibodies, contributes to post-synaptic membrane degeneration in myasthenia gravis.     

实验性变态反应性脑脊髓炎IL-10与IL-18的研究

目的研究IL-10与IL-18在实验性变态反应性脑髓炎(EAE)免疫学发病机制中的变化与作用。方法应用豚鼠诱导Wistar大鼠EAE模型,以豚鼠髓鞘碱性蛋白(MBP)与弗氏完全佐剂(CFA)免疫Wistar大鼠,在第11、18、25d处死大鼠,并采用酶联免疫吸附试验(ELISA)测定IL-10与IL-18水平。结果EAE组的IL-10水平在疾病缓解期升高,IL-18的水平随着疾病的进展逐渐升高,在缓解期有所下降,但均显著高于对照组。结论IL-10与IL-18在EAE的免疫学发病机制中具有重要的作用。     

Increase in anti-astrocyte antibodies in the serum of guinea pigs during active stages of experimental autoimmune encephalomyelitis.

From previous studies on the induction and treatment of experimental autoimmune encephalomyelitis (EAE) in guinea pigs and mice, antibodies have been implicated during both demyelination and remyelination. In the present study, sera from guinea pigs with acute, chronic and myelin basic protein/galactocerebroside (MBP/GC)-treated chronic EAE were evaluated for the presence of anti-glial cell antibodies by immunocytochemical techniques. Antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The majority of sera from acute and chronic active EAE animals displayed intense labelling of astrocytes and only weak staining of oligodendrocytes when tested on sections of normal guinea pig brain tissue. In contrast, sera from animals with chronic EAE treated with MBP/GC gave strong labelling of oligodendrocytes and only minor staining of astrocytes. By immunoblotting, astrocyte staining was shown to be due to the presence of antiglial fibrillary acidic protein (GFAP) antibodies. The intense oligodendrocyte staining observed in sections reacted with sera from MBP/GC-treated guinea pigs corresponded well with high titers of serum anti-GC and anti-MBP antibodies measured by an ELISA. It was concluded that the presence of antibodies against astrocytes was possibly related to astrocytic antigens within the disease-inducing emulsion, at least during the initial phases of EAE, and not to their release from the central nervous system of affected animals.