Genetic and immunological control of human myasthenia gravis

Abstract

Myasthenia gravis (MG) is a classical autoimmune disease characterized by muscle weakness due to an immune attack against the nicotinic acetylcholine receptor (AChR) on the post-synaptic membrane of the neuromuscular junction. Because of the involvement of multiple factors in the disease, the overall aims of this thesis were to uncover the regulation of candidate genes and the possible cellular mechanisms involved in the immuno-regulation of human MG.

MG patients showed some aberrations of the beta2-adrenergic receptor (beta2-AR), such as decreased density of beta2-AR on peripheral blood mononuclear cells (PBMC), serum antibodies against beta2-AR and aberrant distribution of SNPs of codon region within the 02-AR gene. I investigated the correlation of the promoter SNPs of the beta2-AR gene with MG in the present study. I identified a low frequency of allele -468G, -367C, -47C and -20C in MG with thymoma, which confirms the notion that the pathogenesis of MG with thymoma appears to be different from that of other subgroups of the disease. The allele frequency of 468G, -367C, -47C and -20C was increased in MG patients with elevated levels of anti-AChR antibodies and severe generalized disease. It has been shown that this allele could significantly reduce transcription activity, resulting in a decreased density of the beta2-AR on lymphocytes. Therefore, the finding provides an explanation at the molecular level for the decreased density of beta2-AR on patients' lymphocytes.

The pro-inflammatory cytokines, chemokines and their receptors are considered to be important for the localization of immune cells. The chemokine receptor CCR2-64I (G/A) allele is associated with autoimmune diabetes in children whereas CCR5delta32 (32 bp deletion) is correlated to severity of rheumatoid arthritis and prolonged renal allograft survival. In the second study, the association of genetic polymorphisms of the chemokine receptors CCR2 and CCR5 with MG was investigated. The results revealed no association between allelic frequencies of CCR2-641 and CCR5-delta32 and MG.

The cytolytic T lymphocyte-associated antigen-4 (CTLA-4) is a costimulatory receptor expressed mainly on activated T cells and mediates a negative regulation of T cell activation. The genotype G/G at +49 is in strong linkage with -318C/T and is more frequently present in MG patients with thymoma. MG patients carrying -318C showed significantly decreased expression of CTLA-4 mRNA in non-stimulated cells. In the present study, the relationship of the expression of Ctla-4 with the promoter -318C/T SNP was studied in an in vitro system. Two constructs with a reporter gene driven by the Ctla-4 promoter that contained either C or T alleles, were made and transfected in a Jurkat T cell line. The -318T allele showed higher promoter activity than the -318C allele, which confirmed the regulatory effect of this -318C/T SNP.

OX40 (CD 134), a member of TNFR family, is a cell surface glycoprotein expressed on activated CD4+ T cells. Engagement of OX40 by its ligand enhances cytokine production by CD4+ T cells as well as the proliferation and the long-term survival of CD4+ T cells. In the present study, the expression of OX40 on CD4+ T cells from patients with MG was investigated. Results from thirty-six MG patients and twenty-eight healthy controls revealed that MG patients had more CD4+OX40+ T cells in freshly isolated PBMC than healthy individuals. These CD4+OX40+ T cells expressed CD25, but not CD69 and CTLA-4, implying that these T cells are not regulatory cells. High percentages of CD4+OX40+ T cells were found in patients with generalized disease, disease onset at an early age, thymic hyperplasia and high levels of antiAChR antibodies. Upon activation by various concentrations of anti-CD3 antibodies, CD4+ T cells from MG patients showed a tendency towards higher levels of OX40 expression than cells from healthy individuals.

In summary, the association of beta2-AR to MG patients with thymoma confirmed that these patients constitute a separate entity. The down-regulation of beta2-AR in patients with MG, at least partially caused by genetic variants, might up-regulate the expression of CD25 on T cells and promote the expansion of autoreactive T and B cells. The up-regulation of OX40 and down-regulation of CTLA-4 lead to abnormal activation of T cells, especially the auto-reactive T cells. All of these abrrations can eventually contribute to the production of autoantibodies, the abnormal thymic histopathology and disease development in MG.