Treatment of passively transferred experimental autoimmune myasthenia gravis using papain

Abstract

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.

Fig. 1

Clinical course of passive experimental autoimmune myasthenia gravis (EAMG) in groups of three rats receiving single i.p. injections of increasing amounts of mAb35. Points represent mean clinical scores. ◊, 0·05 mg mAb35; ▪, 0·10 mg mAb35; ▴, 0·15 mg mAb35; •, 0·30 mg mAb35. The difference between all groups was found to be statistically significant (P < 0·05), s.d. were <0·6.

Fig. 2

Clinical course of passive experimental autoimmune myasthenia gravis (EAMG) in rats receiving mAb35 with or without papain-mediated protection. Points represent mean clinical scores. Three groups of six rats were injected intraperitoneally with 0·15 mg mAb35; two of the groups then received 0·75 mg of papain in one or three i.p. doses. Another two groups of three rats received only Ringer's buffer or only 0·75 mg papain. The clinical score was evaluated as 0–5 as described in Materials and Methods (0 = healthy, 5 = dead). ◊, mAb35-treated rats; •, mAb35 + papain-treated rats (0·75 mg as a single injection 5 h after the MoAb); ▪, mAb35 + papain-treated rats (three injections, each of 0·25 mg of papain, at 3, 5 and 7 h after the MoAb); ▴, Ringer's buffer only or papain only. The difference between the two groups that received papain and the one that received mAb35 only was found to be statistically significant (P < 0·02), s.d. were <0·5.

Fig. 3

Change in weight following injection of anti-acetylcholine receptor (AChR) mAb35 with or without papain. Points represent mean percentage change in weight. The groups of animals used were those described in Fig. 2. ◊, mAb35-treated rats; •, mAb35 + papain-treated rats (0·75 mg in one injection 5 h after the MoAb); ▪, mAb35 + papain-treated rats (three injections, each of 0·25 mg of papain, at 3, 5 and 7 h after the MoAb); ▴, Ringer's buffer only. The difference between the mAb35 + papain-treated animals and the other groups was found to be statistically significant (P < 0·02), s.d. were <1·6.

Fig. 4

Serum anti-acetylcholine receptor (AChR) antibody concentrations in the groups of rats of Fig. 2. The animals used were those in Fig. 2. ◊, mAb35-treated rats; •, mAb35 + papain-treated rats (0·75 mg in one injection 5 h after the MoAb); ▪, mAb35 + papain-treated rats (0·25 mg papain in three injections 3, 5 and 7 h after the MoAb). The difference between the two groups of mAb35 + papain-treated animals and the one mAb35-treated was found to be statistically significant (P < 0·05).

Fig. 5

Muscle acetylcholine receptor (AChR) content of treated rats. Three groups of six rats were used. Group 1 received only Ringer's buffer, group 2 were injected with 0·15 mg of mAb35 followed by 0·75 mg papain 5 h later, and group 3 were injected only with 0·15 mg of mAb35. Their symptoms were observed at 24 h, then the animals were killed immediately. AChR-containing muscle extracts were prepared from the hind limbs and their AChR content measured. The results are expressed as the muscle AChR content per unit weight of muscle in rats treated with MoAb or MoAb + papain as a percentage of that in rats receiving Ringer's buffer only. Insert: clinical symptoms at 24 h. The difference between the two groups of rats, mAb35-treated and mAb35 + papain-treated, was found to be statistically significant (P < 0·05).