ICOS is essential for the development of experimental autoimmune myasthenia gravis.

Lymphocyte costimulation via the inducible costimulatory molecule (ICOS) is required for effective humoral immunity development. Following immunization with Torpedo acetylcholine receptor (AChR), ICOS gene knockout (KO) mice were highly resistant to clinical experimental autoimmune myasthenia gravis (EAMG) development, had less serum AChR-specific immunoglobulins (Igs), and exhibited a diminutive germinal center (GC) reaction in secondary lymphoid tissues. Lymphocyte proliferation and both Th1 and Th2 differentiation in response to AChR and the AChR dominant alpha146-162 peptide were inhibited by the ICOS gene deficiency. ICOS-mediated lymphocyte costimulation is thus vital to the induction of T cell-mediated humoral immunity to AChR and the development of clinical EAMG.

Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis.

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.

IL-1 receptor antagonist-mediated therapeutic effect in murine myasthenia gravis is associated with suppressed serum proinflammatory cytokines, C3, and anti-acetylcholine receptor IgG1.

In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

Circulating immune complexes augment severity of antibody-mediated myasthenia gravis in hypogammaglobulinemic RIIIS/J mice.

Experimental autoimmune myasthenia gravis (EAMG) is severe in RIIIS/J mice, despite a significant B cell immunodeficiency and a massive TCR V beta gene deletion. Severity of EAMG in RIIIS/J mice is greater than MHC-identical (H-2(r)) B10.RIII mice, suggesting the influence of non-MHC genes as an EAMG-potentiating factor in this strain. To delineate the role of deleted TCR V beta genes in RIIIS/J mice, we obtained (RIIIS/J x B10.RIII)F(1) (V beta(b/c)) x RIIIS/J (V beta(c)) backcross mice using Mendelian genetic methods and immunized them with acetylcholine receptor. EAMG susceptibility was not elevated in mice with V beta(c) genotype having 70% V beta gene deletion. Next, we performed microarray analysis on 12,488 spleen cDNAs obtained from spleens of naive RIIIS/J and B10.RIII mice. In RIIIS/J mice, 263 cDNAs were overexpressed and 303 cDNAs were underexpressed greater than 2-fold, compared with B10.RIII mice. TCR gene expression was augmented, whereas NK receptor, C1q, and C3 gene expressions were diminished in RIIIS/J mice. RIIIS/J mice also had increased lymph node T cell counts, elevated serum anti-AChR Ab levels, and serum C3 and C1q-conjugated circulating immune complex levels. A direct correlation between increased serum C1q-conjugated circulating immune complex levels and disease severity was observed in RIIIS/J mice.

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis.

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.