Direct proof of the in vivo pathogenic role of the AChR autoantibodies from myasthenia gravis patients

Abstract

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.

Figure 1. Clinical course of rats 24 and 48 h post-injection with MG sera and their derivatives.

The figures show the clinical symptoms of groups of rats injected i.p. with the shown volumes of MG sera (or Ig), anti-α/β depleted sera (or Ig) and isolated autoAbs. A–B: EAMG-MG1; C–D: EAMG-MG2; E–F: EAMG-MG3; G: (EAMG-MG4). Control groups were injected with NHS and PBS. Disease severity was assessed on the basis of weakness and scored by their ability to grasp, hang and run when provoked: 0–4 (0: no clinical symptoms; 4: dead, see Methods). Points represent mean clinical scores ±SE.

Figure 2. Weight change of rats treated with MG sera and their derivatives.

The figures show the percent weight change of groups of rats injected i.p. with the shown volumes of MG sera, anti-α/β depleted sera and isolated autoAbs. Control groups were injected with NHS and PBS. Bars represent weight changes (% of controls) +SE, *p<0.05, **p<0.01.

Figure 3. Competition RIA between MG1 autoAbs and ¹²⁵I-α-Bgtx for binding to the AChR.

Anti-AChR Ab precipitation of MG1 serum by RIA and competition RIA for the detection of blocking Abs in the serum. For this, unlabeled AChR was preincubated with MG1 serum followed by the subsequent addition of ¹²⁵I-α-Bgtx (MG before ¹²⁵I-α-Bgtx); in parallel AChR was preincubated with ¹²⁵I-α-Bgtx followed by the subsequent addition of MG1 (MG after ¹²⁵I-α-Bgtx). The precipitated radioactivity, after the addition of the anti-Ab, was equal in both assays, indicating that no autoAbs of MG1 bind near or at the acetylcholine binding site.

Figure 4. Muscle AChR content of treated rats.

The four groups of animals injected with MG sera (MG1–MG4) and their derivatives were sacrificed 48 hours post-injection. The hind limb muscles of each rat were dissected and the extracted AChRs were estimated by RIA. Results are expressed as a percentage of the AChR of the rats receiving NHS only. Results for PBS are not shown but were very similar to the NHS. a, b, c, d: rats injected with sera and derivatives of MG1, MG2, MG3 and MG4 respectively. Bars represent mean AChR content (% of controls) +SE, *p<0.05, **p<0.01.

Figure 5. Deposition of IgG at the NMJ of EAMG rats detected by confocal microscopy.

Hind limb muscle specimens were obtained from the rats injected with MG1 and MG3 sera and derivatives as well as from rats injected with NHS. Cryostat muscle sections were co-stained with Alexa Fluor 555-α-Bgtx to identify AChR (red) and with F(ab2) donkey anti-human IgG FITC conjugated to identify human autoAbs (green); merge on the right. The immunofluorescence data shown are representative of sections obtained from two rats of each group. Original magnification is 350×.