Immunoregulation in experimental autoimmune myasthenia gravis

Abstract: Myasthenia gravis (MG) derives its name from Latin and Greek words meaning, "grave muscle weakness". MG is a classical T cell dependent antibody-mediated autoimmune disease. Experimental autoimmune myasthenia gravis (EAMG), representing an animal model of human MG, can be induced in susceptible animals by immunization with acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). EAMG has clinical, pharmacologic and electrophysiologic properties similar with those of human MG. Studies on EAMG have helped to understand mechanisms of immunoregulation and provided information on treatment of patients with MG. In MG and EAMG, the major event of pathogenesis can be attributed to the immune attack by autoantibodies against AChR at the endplate of the neuromuscular junctions. T cell abnormalities in MG and EAMG facilitate anti-AChR antibody (Ab) production by means of cell-cell contact and locally secreted cytokines. The aims of this study are to identify roles of immunoregulatory cells (CD4+ Th3 cells, NK1.1+ cells) and other immunoregulatory elements such as cytotoxic T lymphocyte antigen-4 (CTLA-4), and cytokines/cytokine receptors in the pathogenesis of MG. CTLA-4 plays critical roles in downregulating T cell activation and maintaining lymphocyte homeostasis. Anti-CTLA-4 Ab treatment in vivo enhances host anti-tumor immunity and exacerbates T cell-mediated autoimmune diseases. We demonstrate that CTLA-4 blockade enhances T cell proliferative responses to AChR and anti-AChR Ab production, and provokes rapid onset and increased severity of EAMG in C57BL/6 (B6) mice. CTLA-4 blockade also provokes development of MG after immunization of B6 mice with the immunodominant AChR peptide [alpha]146-162, at least in part by triggering epitope spreading. Finally, we demonstrate that the effects observed after CTLA-4 blockade are not limited to the initial phase of EAMG. The notion that NK cells may affect the outcome of adaptive immune responses is tested in murine MG. We show that depletion of NK1.1+ cells (NK and NKT cells) prior to immunization with AChR in CFA inhibits anti-AChR Ab production and the development of MG in B6 mice, which is associated with a failure to mount Th1 responses. We confirm this finding by demonstrating that IL-18 knockout (IL-18-/-) mutant, who are deficient in NK cell activity, are resistant to the induction of MG. In contrast, CD1d1-/- and J[alpha]281-/- (both are deficient in J[alpha] cells) mutants develop EAMG to a similar extent as wild type J[alpha] mice. We show that nasal administration of AChR in B6 mice, before immunization with AChR in J[alpha] results in delayed onset and reduced muscle weakness compared with control mice. These effects are associated with decreased AChR-specific lymphocyte proliferation and decreased levels of anti-AChR Abs of the IgG2a and IgG2b isotypes in serum. To address the mechanisms underlying nasal tolerance in murine MG, we compare clinical and immunological parameters in the AChR-pretreated wt mice and J[alpha]CD8-/- mice. AChR-pretreated wt and CD8-/- mice are similary protected from EAMG and show augmented TGF-[beta] and reduced IFB-[gamma] responses. Splenocytes from AChR-pretreated wt and CD8-/- mice, but not from CD4-/- mice, suppress AChR-specific T cell proliferation that can be blocked by Abs against TGF-[beta]. To evaluate the role for IFN-[gamma] in the pathogenesis of EAMG, we injected recombinant rat IFN-[gamma] (rrIFN-[gamma]) at the time of immunization with AChR in CFA to EAMG-susceptible Lewis rats, and EAMG-resistant Wistar Furth (WF) rats. RrIFN-[gamma] enhances Lewis rat EAMG. The exacerbated muscular weakness is associated with higher levels of anti-AChR IgG and enhanced expression of TNF-[alpha] by mononuclear cells. Anti-AChR IgG Ab levels in WF rats are augmented to a similar extent as in Lewis rats, however, the identical procedure induced only mild and transient EAMG in WF rats due to inherent low Th1 responses and default Th3 phenotype development. Tumor necrosis factor receptor-1 (TNFR1) has been implicated in the pathogenesis of T cell- mediated autoimmune disorders, but its role in Ab-mediated autoimmune diseases has not been addressed. We show that TNFR1-/- mice fail to develop EAMG. Mononuclear cells from TNFR1-/- mice produce low amounts of Th1 (IFN-[gamma], IL-2, IL-12)-type cytokines, but increased levels of Th2 (IL-4, IL- 10)-type cytokines compared with those from wt mice. The levels of anti-AChR IgG2 Abs are strongly reduced and those of anti-AChR IgG1 Abs are moderately reduced. Co-injection of recombinant mouse IL-12 (rmIL-12) with AChR in adjuvant restores T cell responses to AChR and promotes development of EAMG in TNFR1-/- mice. We conclude that 1) A process of intramolecular determinant spreading, leading to diversification of anti-AChR Ab repertoire, is one mechanism responsible for the exacerbation of EAMG after CTLA-4 blockade. 2) NK cells, but not NKT cells, determine the outcome of EAMG via control of autoreactive T and B cells. 3) AChR-specific CD4+ Th3 cells are identified as regulatory cells that are responsible for the establishment of tolerance against AChR in B6 mice. 4) IFN-[gamma] plays a major role in the pathogenesis of EAMG in the Lewis rat, but fails to break disease resistance in the WF rat. 5) TNFR1 is critically involved in the development of EAMG. However, the absence of TNFR1 can be compensated by addition of IL-12 at least at the priming stage of EAMG. This finding provides a plausible mechanism of counteraction of various cytokine pathways in MG.