Changes in inflammatory cytokine networks in myasthenia gravis

Myasthenia gravis (MG) is an autoimmunological inflammatory disorder of the neuromuscular junction. Inflammation could be a key player for understanding the pathogenesis of MG. We measured the serum levels of 24 inflammatory cytokines in 43 patients with anti-acetylcholine receptor antibody-positive MG and 25 healthy controls. In patients with MG, serum levels of a proliferation-inducing ligand (APRIL), IL-19, IL-20, IL-28A and IL-35 were significantly increased as compared with controls (p < 0.05). Among them, IL-20, IL-28A and IL-35 were significantly decreased after treatment (p < 0.05). In clinical subtype analyses, APRIL and IL-20 were increased in patients with late-onset MG and IL-28A levels were increased in patients with thymoma-associated MG compared with healthy controls (p < 0.01). The results of the present study demonstrate both anti-inflammatory and inflammatory cytokines are upregulated in MG, reflecting the importance of cytokine-mediated inflammation and its regulation in MG pathophysiology.


Materials and Methods
Subjects. Forty-three Japanese patients with anti-AChR antibody positive MG and 25 healthy participants of similar age and sex (men, 9; women, 16; mean age, 52.4 years; range, 33-72 years) were included in the present study. We reviewed patient data regarding sex, age, disease duration, anti-AChR antibody titer, E-L-T classification (early-onset MG [EOMG], age at onset ≤ 49 years; late-onset MG [LOMG], age at onset ≥ 50 years; thymoma-associated MG [TAMG]) 8 , MG Foundation of America (MGFA) classification 9 and quantitative MG (QMG) scores 9 at the time of serum sampling. No patients with MG received immunosuppressive therapy then. Serum samples were obtained from 10 patients with MG after initiation of immunosuppressive therapy.
Ethical approval was granted by the Ethics Committee of the Chiba University School of Medicine, Chiba, Japan and all experiments were performed in accordance with relevant guidelines and regulations. All subjects provided written informed consent for their participation in the present study.
Significant correlations were observed between serum levels of APRIL, IL-19, IL-20, IL-28A and IL-35 (Table 2). No significant correlation was observed between clinical manifestations of MG and the serum levels of the above-described cytokines (data not shown). Cytokines, in addition to anti-AChR antibodies and complement proteins, play a major role in the development of inflammation at the neuromuscular junction in MG. A previous study in an experimental autoimmune myasthenia gravis model demonstrated that increased proportion of Th1 (type 1 helper T cell) and Th17 cells worsen MG pathogenesis, whereas increased proportions of Th2 and Treg (regulatory T cell) cells ameliorate MG development 10 . Similar results have been reported in studies of patients with MG, including the upregulation of IL-17 levels 6 and downregulation of IL-4 levels 3 .  has been shown to activate immune cells, stimulate the release of pro-inflammatory cytokines (e.g., IL-6 and TNF-α ) 12 and upregulate Th2 responses 13 . Conversely, IL-19 knockout mice have recently been reported to develop exacerbations of colitis 14 and IL-19 deficiency has been shown to increase the production of pro-inflammatory cytokines in activated microglia 15 , indicating the immunopathological relevance of IL-19 as an anti-inflammatory cytokine. IL-20 acts as a proinflammatory cytokine and has been shown to be upregulated in the synovial fluid of patients with rheumatoid arthritis 16 and in the psoriatic lesions 17 and to be associated with lupus nephritis 18 . In addition, IL-19 and IL-20 enhance tissue remodeling activities and angiogenesis in response to inflammation 19 . VEGF, which is an important cytokine in the promotion of angiogenesis, has also been shown to be upregulated in MG 3 . Elevations in the serum levels of these cytokines in MG may reflect the presence of angiogenesis in response to inflammation. Further studies are required to validate the functions of these cytokines in MG pathogenesis.
IL-28A is known as a type III interferon and has potent antiviral effects. IL-28 promoted Th1 skewing and inhibited Th2 and Th17 responses in an allergic asthma model 20 and IL-28-treated dendritic cells induced the proliferation of Treg cells 21 . Therefore, elevated IL-28 levels may have a protective role in MG pathogenesis. Regarding other IL-10 family cytokines, IL-10, which is a representative anti-inflammatory cytokine and IL-22 are reportedly downregulated in patients with MG. In patients with MG, B cell-derived IL-10 is downregulated 22 , whereas serum IL-10 levels tended to be upregulated 3 . Serum IL-22 levels were downregulated and negatively correlated with anti-AChR antibody titers in patients with MG 7 . These results indicate IL-10 family cytokines have multivalent functions and may play essential roles in mechanisms underlying the development of inflammation in MG.
IL-35, which is a member of the IL-12 family, is produced by the Treg cells and functions as an anti-inflammatory cytokine 23 . Although no previous studies have demonstrated an association between IL-35 and the development of MG, the results of the present study indicate IL-35 may perform anti-inflammatory and protective functions in MG.
APRIL, which is a member of the TNF-ligand superfamily, is secreted by macrophages, T cells and dendritic cells and has an important role in the maturation and survival of B cells 24 . B cells are responsible for the production of autoantibodies against AChR in MG. A previous study reported no difference in APRIL and BAFF levels between patients with MG and controls 25 . Thymic expression of APRIL and BAFF may represent a source of these cytokines in MG. Interestingly, APRIL-positive cells have been confirmed in hyperplastic thymus and atrophic thymus but were scant in thymoma 25 , which may explain the difference in APRIL levels between EOMG, LOMG and TAMG. The results indicate that the upregulation of APRIL in MG may influence the production of autoantibodies by B cells.
The present study had some limitations. The sample size of patients with MG, especially in the MG subgroups, was relatively small, which may limit the generalisability of the present study. We were unable to demonstrate significant correlations between serum cytokines levels and clinical parameters. Further, serum cytokines levels may not necessarily reflect those levels at neuromuscular junction. Further studies are required to address these limitations.
In conclusion, the results of the present study demonstrate significantly increased serum levels of pro-and anti-inflammatory cytokines in patients with MG. Several inflammation-related cytokines may form complicate networks and play an important role in mediating pathogenic and inflammatory mechanisms at the neuromuscular junction in the pathogenesis of MG. Greater understanding of cytokine signaling pathways may facilitate the development of novel treatments for MG.