Myasthenia gravis, (MG) is an autoimmune disorder characterized by muscle weakness. MG patients have impaired neuromuscular transmission caused by antibodies reactive with the nicotinic acetylcholine receptor (nAChR). Based on clinical, pathologic, and immunologic lines of evidence, the thymus has long been considered to play a pivotal role in the pathogenesis of this disease. The thymus in MG is an anomalous site of B cell activity. Germinal center (GC) hyperplasia is seen in greater than 75% of patients. The recovered B cells have properties consistent with antecedent in vivo activation and they secrete the autoantibody anti-AChR. AChR expressed in the thymus may play an important role in the autosensitization process or in the breakdown of tolerance mechanisms that normally prevent the expression of autoimmunity. The overall goal of this project is to enhance our understanding of the immunologic and molecular properties of thymic B cells and thymic nAchRalpha.
Specific aims are to: l) determine the functional and molecular properties of MG thymic GC B cells 2) determine VH and VL gene utilization of thymic anti-AChRalpha antibodies, and 3) further characterize nAChR alpha chain (nAChRalpha) expressed in normal and myasthenic human thymus with regard to a) protein expression, b) quantitative mRNA c) tissue localization of mRNA, d) regulation of mRNA by cytokines e) ontogeny of mRNA expression, and l) the capacity of thymic nAChR to induce specific immunologic unresponsiveness.
These aims will be approached, in part, by using thymic and blood cells obtained at thymectomy of MG patients and cardiac surgery of control subjects. One hypothesis to be tested is that anti-AChR is a product of GC B cells. We will also test the hypothesis that thymic GC B cells will show the molecular features of antigen stimulation and show skewed V-gene utilization if GC's are induced by a common pathologic event. A filamentous phage system will be used to clone and sequence anti-AChRalpha VH and VL genes used by thymic B cells. Neonatal mice will undergo intrathymic injection with AChR-bearing cells to determine if introduction of AChR into the thymus can induce unresponsiveness to this autoantigen when it is subsequently administered in an immunogenic manner. We will also carry out an analysis of the ontogeny of thymic AChRalpha expression to determine if autoreactive T cells might escape prior to expression of thymic AChR. Quantitative and qualitative features of thymic nAChRalpha will be determined using a recently developed quantitative RT-PCR and in situ hybridization. These findings are of great potential clinical importance since anti-AChR antibody is pathogenic in MG and thymectomy is often beneficial in this disorder.
