Regulatory T cells induced by GM-CSF suppress ongoing experimental myasthenia gravis
Introduction
Myasthenia gravis (MG) is an antibody-mediated autoimmune disease in which pathogenic autoantibodies are directed against the skeletal muscle nicotinic acetylcholine receptor (AChR) [1]. Experimental autoimmune myasthenia gravis (EAMG) induced in C57BL/6 mice after repeated immunizations with emulsified Torpedo californica AChR (tAChR) is a useful model for the study of pathogenic mechanisms and therapeutic strategies relevant to MG in humans [2]. Although antibodies to the AChR are directly responsible for the destruction of the muscle endplate resulting in both MG and EAMG, the autoantibody response is T cell dependent, with CD4+ T cells providing help for B cells to produce anti-AChR antibodies [3], [4]. Despite the fact that the target antigen is so well defined, there is currently no specific immunosuppressive therapy or cure for MG. Nonspecific immunotherapy utilizing corticosteroids and other immunosuppressive drugs combined with symptomatic therapy with acetylcholinesterase inhibitors results in clinical improvement and substantial control of symptoms in most patients. However, there are significant potential side effects and risks associated with global nonspecific suppression of the immune response, including infections and malignancy.
Ideal specific therapies for MG would have little effect on overall immunity, while targeting the mechanisms that initiate and sustain the autoimmune response to the AChR. While these mechanisms are not completely understood, multiple lines of evidence indicate that the immune system's professional antigen-presenting cells, the dendritic cells (DCs), participate in the onset and progression of autoimmune diseases [5], [6]. Animal models show that the transfer of DCs isolated from donors with acute autoimmune disease or propagated in vitro under conditions that induce maturation, generates a strong T helper (Th)-1 response, eventually culminating in autoimmune disease [7]. Conversely, DCs have been shown to have the ability to educate T cells to tolerate self antigens, and to promote the mobilization of regulatory T cell (Treg) subsets [8], [9], [10]. It has been shown that the interaction of DCs with antigen-specific Tregs can suppress experimental autoimmunity [11]. Current evidence indicates that the immunogenic or tolerogenic function of DCs is largely determined by differentiation status which may be manipulated using growth factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF) [12], and that DC functional state is important in determining Treg biology and antigen-specific control of experimental autoimmunity [13], [14]. Previous work has been published examining the potential of in vivo administration of GM-CSF in experimental autoimmune thyroiditis (EAT) [15], [16], and in the experimental model of autoimmune diabetes [14], and mobilization of specific DC subsets and Tregs was reported to critical to the observed effects. But, EAT and autoimmune diabetes are T-cell mediated diseases, and in general, the role of dendritic cells (DCs) in the biology of regulatory T cells and subsequent control of autoimmunity has been studied primarily in T cell mediated autoimmune diseases. We, however, have previously observed that GM-CSF had profound effects on the induction of experimental autoimmune myasthenia gravis (EAMG), a well-characterized antibody-mediated autoimmune disease [17].
In the current study, we examine the therapeutic potential of GM-CSF in chronic EAMG, and demonstrate that GM-CSF effectively ameliorates clinical disease in mice with ongoing, well-established disease. Furthermore, we show not only an effect of GM-CSF on particular subpopulations of DCs, T cells, and T cell proliferative response to the AChR, but also a significant down-modulation of pathogenic anti-AChR autoantibody production.
Section snippets
Mice
Eight-week old female C57BL6/J mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were housed in the Biologic Resources Laboratory facilities at the University of Illinois (Chicago, IL) and provided food and water ad libitum. All mice were cared for in accordance with the guidelines set forth by the University of Illinois Animal Care and Use committee.
Purification of tAChR and mouse AChR
Torpedo AChR (tAChR) was purified from the electric organs of T. californica by affinity chromatography using a conjugate
GM-CSF treatment effect in established EAMG
At the end of the disease induction (initial priming immunization and 3 boosters) stage, greater than 90% of the animals had clinically demonstrable fatigable muscle weakness. We then divided these animals into two groups (n = 20) with an equal balance of the various disease severities in each group. We treated one group of animals with GM-CSF (2 µg IP daily for 10 days) and the other group of animals with PBS. The day of treatment initiation was designated “day 0” and the mice were clinically
Discussion
In this study, we expanded upon our previous observations that treatment with GM-CSF protected against the induction of EAMG [17], by investigating the therapeutic potential of GM-CSF in the treatment of ongoing, active EAMG, a well-characterized antibody-mediated autoimmune disease. We found that treatment with GM-CSF at a stage of chronic, well-established disease, effectively induced improvement in signs of muscle weakness and potently suppressed disease progression. Understanding the
Acknowledgments
This work was supported by the NIH (National Institute of Neurologic Disorders and Stroke, K08NS058800-01, MNM; and National Institute of Allergy and Infectious Diseases, RO1 AI 058190-01, BSP); and by the Myasthenia Gravis Foundation of America — Postdoctoral Fellowship Award to JRS.
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