RESUMO
Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-γ, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-γ and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients differentially.
Assuntos
Autoanticorpos/imunologia , Citocinas/sangue , Miastenia Gravis/sangue , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Adulto , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/genética , Ligante de CD40/metabolismo , Separação Celular , Demografia , Feminino , Humanos , Terapia de Imunossupressão , Subunidade p40 da Interleucina-12/sangue , Leucócitos Mononucleares/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Myasthenia gravis (MG) is an autoimmune disorder affecting the neuromuscular junction, usually caused by autoantibodies against the acetylcholine receptor (AChR) or the muscle-specific kinase (MuSK). Our aim is the development of a therapy based on the selective extracorporeal elimination of anti-AChR or anti-MuSK antibodies. To this end, the extracellular domains of the AChR subunits and MuSK have been expressed in yeast to be used as adsorbents, after optimization, and to obtain large quantities of proteins with near-native structure. We have characterized these proteins with respect to their use as specific immunoadsorbents for MG autoantibodies, and have begun large-scale experiments in order to verify the feasibility of application of the method for therapy. Furthermore, we have initiated animal studies to test possible toxicity and safety issues of the adsorbents or the procedure itself. The successful completion of the scale-up and safety tests will allow the initiation of clinical trials.
