Interferon gamma (IFN-gamma) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice

Abstract

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) (+/+) mice of H-2(b) haplotype with AChR in CFA. We observed that AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its dominant pathogenic alpha146-162 sequence when compared with these cells from the WT mice. However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a antibodies. However, keyhole limpet hemocyanin (KLH)-priming of IFN-gko mice readily elicited both T cell and antibody responses, suggesting that IFN-gamma regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that IFN-gamma is required for the generation of a pathogenic anti-AChR humoral immune response and for conferring susceptibility of mice to clinical EAMG.

Figure 1

(A) Effect of IFN-γ gene disruption on in vitro lymphocyte proliferation in response to AChR (10 μg/ml) and control antigen (OVA; 20 μg/ml) on day 5 after immunization with 20 μg AChR in CFA. Results were expressed as a stimulation index. Background cpm in the absence of antigen for C57BL/6, WT, and IFN-gko mice are as follows: 1,019, 1,039, 3,443. (B). Effect of IFN-γ gene disruption on in vitro lymphocyte proliferation in response to KLH (50 μg/ml) and control antigen (OVA; 20 μg/ml) on day 5 after immunization with 100 μg KLH in CFA. Background cpm in the absence of antigen for C57BL/6, WT, and IFN-gko mice are as follows: 15,184, 14,223, 17,095.

Figure 2

RIA of serum anti-AChR Ab to mouse muscle AChR. Serum samples were collected on day 89 after immunization. The difference in anti-AChR antibody levels between WT and IFN-gko mice (P = 0.002) and between C57BL/6 and IFN-gko mice (P = 0.003) was statistically significant. The difference in anti-AChR antibody levels between WT and C57BL/6 mice (P = 0.222) is not statistically significant. —*, the mean value.

Figure 3

The effect of IFN-γ gene disruption on anti-AChR Ab isotypes (A) IgG1, (B) IgG2a and anti-KLH Ab isotypes, (C) IgG1, (D) IgG2a. Serum samples were collected on day 89 after the first immunization with AChR or KLH. The differences in IgG1 and IgG2a responses between AChR-immunized WT and IFN-gko mice were statistically significant; for IgG1 response, P = 0.000; for IgG2a response, P = 0.000. *, the mean value.

Figure 4

Hypothetical model: how IFN-γ influences the humoral immune response to self (AChR) and foreign (AChR) antigens in vivo.