Alicia L. Degano

Passive transfer of experimental autoimmune encephalomyelitis in Wistar rats: dissociation of clinical symptoms and biochemical alterations.

We have used passive transfer of myelin‐reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freunds adjuvant‐immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen‐specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE‐transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease. J. Neurosci. Res. 59:283–290, 2000

Inhibitory role of diazepam on autoimmune inflammation in rats with experimental autoimmune encephalomyelitis.

Glutamate and GABA are the main excitatory and inhibitory neurotransmitters in the CNS, and both may be involved in the neuronal dysfunction in neurodegenerative conditions. We have recently found that glutamate release was decreased in isolated synaptosomes from the rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. In contrast to control animals where GABA induced a decrease in the evoked glutamate release, which was abolished by picrotoxin (a GABA(A) antagonist), synaptosomes from EAE rats showed a loss in the inhibition of the glutamate release mediated by GABA with a concomitant diminution of the flunitrazepam-sensitive GABA(A) receptor density. We have presently further evaluated the relevance of the GABAergic system in EAE by treating rats challenged for the disease with the GABA agonist diazepam. Administration of diazepam during 6 days starting at day 6 or 11 after EAE active induction led to a marked decrease of the disease incidence and histological signs associated with the disease. Cellular reactivity and antibody responses against the encephalitogenic myelin basic protein were also diminished. Beyond the effects of diazepam on the autoimmune, inflammatory response, we report also a positive effect on neurotransmission. Treatment with diazepam inhibited the previously described reduction in glutamate release in the frontal cortex synaptosomes from EAE animals. These data suggest that an endogenous inhibitory GABAergic system within the immune system is involved in the diazepam effect on EAE and indicate that increasing GABAergic activity potently ameliorates EAE.

Interaction between Gonadal Steroids and Neuroimmune System in Acute Experimental Autoimmune Encephalomyelitis (EAE) in Wistar Rats

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T lymphocytes directed against myelin antigens. Since neuroendocrine-immune dysfunction appears to contribute to the pathogenesis of autoimmune diseases, the present work was designed to study the effect of changes in the endocrine system on the development of acute EAE and the immune response against myelin basic protein (MBP). Intact and sham males and intact female Wistar rats showed the most severe clinical symptoms (acute period) 12–14 days post-inoculation (dpi). Then, they began gradually to recover, regaining the total ability to walk by 15–17 dpi. Male Wistar rats with altered levels of gonadal hormones by surgical castration showed an onset of the symptoms retarded 2–3 days with respect to the other EAE groups, showing neuropathological symptoms up to 27–28 dpi, and remaining with lower body weight even at 40 dpi. The castrated animals exhibited a specific delay in MBP-stimulated DTH reactivity that correlates with the delay in the onset of the clinical symptoms. Also significant lymphocyte proliferation to MBP was still present at 35 dpi that was absent in the sham group. The distribution of the IgG subclasses indicated that at 35 dpi castrated animals have a higher IgG2b/IgG1 ratio (35.1) in comparison to that presented by sham rats (4.8). Considering that at this time the castrated animals were not completely recuperated, these results could indicate an ongoing inflammatory immune response associated with Th1 activity in these animals. Also castrated animals developed antibodies to a diversity of MBP epitopes in comparison to sham rats, which presented a dominance of antibodies to MBP peptide p96–128. These results indicate that sex hormones levels regulate cell-mediated immunity and the specificity of anti-MBP antibodies related to the induction and development of acute EAE.

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline‐soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity‐purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen‐driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens. J. Neurosci. Res. 53:187–194, 1998.

Neuronal antigens modulate the immune response associated with experimental autoimmune encephalomyelitis

Rats primed with bovine myelin (BM) in complete Freunds adjuvant, develop acute experimental autoimmune encephalomyelitis (EAE). We have previously described that intraperitoneal administration prior to the active induction of the disease of a bovine synaptosomal fraction (BSF) and BM were effective ways of suppressing EAE. We found that both treatments diminish the incidence of the disease and reduced biochemical and histological alterations of the central nervous system (CNS). To characterize this suppression process, in this study we examined the antigen‐specific immune response in animals protected from EAE. Lymph node mononuclear cells derived from sick EAE rats, as well as from those protected by BM and BSF, showed strong myelin basic protein (MBP) proliferation. Analysis of the humoral response against MBP showed a significant diminution of IgG2b anti‐MBP titres in protected BM and BSF rats in contrast to sick EAE rats whose condition could be related to a diminished anti‐MBP Th1 response. Finally, cells from rats protected by BSF and BM reduced the incidence of EAE when they were adoptively transferred into animals prior to active induction of the disease. These results suggest that a mechanism based on the generation of regulatory cells and immune deviation could account for the EAE suppression mediated by myelin as well as synaptosomal antigens.

Glutamate Release Machinery Is Altered in the Frontal Cortex of Rats with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.

Synapsin-induced proliferation of T-cell lines against myelin basic protein obtained from rats with experimental autoimmune encephalomyelitis.

We have previously described that antibodies and T cells against myelin basic protein (MBP) rose under conditions to induce acute experimental autoimmune encephalomyelitis (EAE) bind other proteins present in the synaptosomal fraction, some of them identified as synapsin I. The aim of this study was to evaluate whether anti-MBP T-cell lines can be also activated by synapsin. The analysis of rat anti-MBP T-cell lines cultured with each antigen showed that these cells responded also to purified rat synapsin and to the amino terminal portion of this protein. This recognition originated a proliferative response with a concomitant pattern of cytokine secretion similar to that induced by MBP itself implicating that this recognition would be mediated by the T-cell receptor. On the other hand, anti-synapsin T-cell lines were not capable of responding to MBP stimulation. Therefore, the immunological cross-reactivity between both proteins occurs only in one direction and these cross-reactive cells would be elicited only in animals sensitized with MBP. A possible implication of immunological agents against MBP cross-reactive with extra-myelin proteins in the process of EAE is considered.

Time course of IgM antibodies which block anti-myelin basic protein IgG antibodies associated with development of experimental autoimmune encephalomyelitis in rabbits

Several authors have demonstrated the presence in normal sera of antibodies that inhibit binding of a variety of autoantibodies. These inhibitory or blocking antibodies are generally considered to play a role in humoral self-tolerance. We examined sera from normal rabbits and from rabbits with experimental autoimmune encephalomyelitis (EAE), in search for antibodies capable to inhibit reactivity of autoantibodies directed to myelin basic protein (MBP). Rabbits injected with bovine myelin in complete Freunds adjuvant (EAE rabbits) or with adjuvant alone (control rabbits) were bled at various intervals post-injection. Sera were subjected to chomatography on a protein A-Sepharose column, retained and nonretained fractions were collected, and ability of these fractions to block reactivity of affinity-purified anti-MBP IgG-antibodies was analyzed by immunoblot technique. Protein A nonretained fraction from control rabbits inhibited anti-MBP IgG reactivity to the same degree at all intervals tested, whereas the same fraction from EAE animals showed an increase in inhibitory activity after induction of the disease. This inhibitory activity declined with the onset of clinical symptoms, and remained low in rabbits that did not recover from the disease. In contrast, the inhibitory activity remained at maximum value in EAE rabbits with spontaneous remission of clinical symptoms. We showed that the inhibitory activity is due to IgM-antibodies, and discussed the role of these antibodies in the development of EAE.