RESUMO
The immuno phenotypic profile of the mononuclear cells that bind myelin basic protein (MBP) and synapsin was investigated in lymph node cells from rats with experimental allergic encephalomyelitis induced by injection with MBP. Using a double immunofluorescent labeling technique, purified cells that bind one or both antigens were analyzed in different stages of the disease. The total MBP-bound lymphocytes increased at 14 days post-inoculation (dpi), had a CD4+/CD8+ ratio of two and were present until 29 dpi. Conversely, the apportionment of cells specific for MBP that also recognize synapsin reached a maximum value at 14 dpi coincidentally with the expression of the paralysis symptoms and then, they disappeared when the animal began to recover. This population amounted to about 40% of the total lymph node MBP-bound cells and had a CD4+/CD8+ ratio of one, indicating that the lymphocytes with MBP-synapsin crossreactivity could be principally implicated in a cytotoxic or suppressor activity.
Assuntos
Doenças Autoimunes/imunologia , Encefalomielite Autoimune Experimental/imunologia , Proteína Básica da Mielina/imunologia , Sinapsinas/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Doenças Autoimunes/patologia , Relação CD4-CD8 , Bovinos , Convalescença , Reações Cruzadas , Encefalomielite Autoimune Experimental/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Imunização , Imunofenotipagem , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Proteína Básica da Mielina/toxicidade , Ratos , Subpopulações de Linfócitos T/patologia , Fatores de TempoRESUMO
A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.
Assuntos
Actinas/metabolismo , Proteína Básica da Mielina/metabolismo , Actinas/química , Animais , Sítios de Ligação , Bovinos , Corantes Fluorescentes , Soros Imunes , Músculos/metabolismo , Proteína Básica da Mielina/química , Naftalenossulfonatos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Coelhos , Espectrometria de Fluorescência , Medula Espinal/metabolismo , Sinapsinas/metabolismoRESUMO
We previously demonstrated that antibodies against myelin basic protein (MBP) obtained from animals with experimental allergic encephalomyelitis (EAE), induced with MBP and purified by affinity chromatography, have the property to recognize a neuronal protein, synapsin Ia and Ib. To investigate whether this crossreactivity also occurs at the cellular level, we purified spleen and lymph node mononuclear cells from rats sensitized with MBP or synapsin using polystyrene plates coated with the respective antigen. We observed that animals injected with MBP have T lymphocytes that bind both antigens. Using the same system, each purified cell population was confronted again to the studied antigens. The anti-MBP cells recognized once more epitopes of MBP and about 40% of them also recognized synapsin. On the other hand, cells that first were attached to synapsin, in the second exposure to antigens bound to MBP and synapsin in similar amounts. Double immunofluorescent labeling of the mononuclear cells isolated from animals injected with bovine myelin or MBP showed that the same lymphocyte was able to recognize MBP as well as synapsin. In both experimental systems the quantitative results were similar indicating that in bovine myelin- or MBP-sensitized animals practically all the cells that recognize synapsin are anti-MBP cells, and of the total cells raised against MBP, around 40% of them show this crossreactivity. On the contrary, animals injected with synapsin have cells that bind to this protein but not to MBP indicating that the described crossreactivity, as observed at humoral level, is only in one way.(ABSTRACT TRUNCATED AT 250 WORDS)