The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice

Abstract

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Fig. 1

Experimental autoimmune myasthenia gravis (EAMG) is markedly exacerbated in Daf1^–/–CD59a^–/– mice. (a) To confirm that the monoclonal antibody (mAb) McAb-3 activated complement (C), a plate assay was established in which the mAb was coated onto enzyme-linked immunosorbent assay (ELISA) plate wells and capacity to activate C from serum assessed by measuring C3b deposited on the plastic. Antibody-coated wells are shown as diamonds and wells without mAb as triangles. Results are means of quadruplicate determinations at each point. Standard errors are plotted but are not visible in this highly reproducible assay. (b–d) EAMG was induced in age- and sex-matched groups of five wild-type (diamonds), Daf1^–/–(squares), Cd59a^–/– (triangles) and Daf1^–/–CD59a^–/– (circles) mice. Weight (b), grip strength (c) and clinical score (d) were measured at intervals. Data are means ± standard deviation (s.d.) (n = 5) for each point. Weight was reduced significantly compared to controls (P < 0·05) only in Daf1^–/–Cd59a^–/– mice from 24 h onwards. Grip strength was reduced significantly compared to controls in Cd59a^–/– mice at 24, 38 and 48 h (P < 0·05) and Daf1^–/–Cd59a^–/– mice at all time-points (P < 0·01). Clinical score was increased significantly compared to controls in Daf1^–/– and Cd59a^–/– mice at 38 and 48 h (P < 0·05) and in Daf1^–/–Cd59a^–/– mice (P < 0·05 at 18 and 24 h, P < 0·01 at 38 and 48 h).

Fig. 2

Inhibition of C5 cleavage protects from disease in Daf1^–/–CD59a^–/– mice. In a separate experiment, experimental autoimmune myasthenia gravis (EAMG) was induced in two age- and sex-matched groups of five Daf1^–/–CD59a^–/– mice. One group was given anti-C5 monoclonal antibody (mAb) with the disease-initiating antibody (squares), while the other group was given initiating antibody alone (diamonds). Weight (a), grip strength (b) and clinical score (c) were measured at intervals. Data are means ± standard deviation (s.d.) (n = 5) for each point. Differences between treated and untreated groups for each of the three measured parameters were significant (P < 0·05 to P < 0·01) at all time-points post-induction.

Fig. 3

Acetylcholine receptor (AChR) loss is exacerbated in Daf1^–/–CD59a^–/– mice. Muscle sections were stained with α-bungarotoxin–rhodamine and imaged. Twenty fields were captured for each muscle analysed. Representative fields for wild-type (a), Daf1^–/–CD59a^–/– (b), Cd59a^–/– (c) and Daf1^–/– (d) mice are shown. Plate (e) is a representative section for Daf1^–/–Cd59a^–/– mice treated with anti-C5 monoclonal antibody (mAb). Arrows indicate positive areas. Magnification × 200. The area occupied by α-bungarotoxin-positive end-plates was measured automatically using the density-slicing feature of the Openlab software and the mean value from 20 fields for each mouse calculated. (f) Mean of these means ± standard deviation (s.d.) (n = 5) for the first experiment comparing each of the four mouse strains used; end-plate area was reduced significantly in Daf1^–/–Cd59a^–/– mice compared to all other groups (only comparisons with wild-type are shown for clarity). (g) Means of means for the second experiment, testing effects of C5 inhibition in Daf1^–/–Cd59a^–/– mice; end-plate area was preserved by anti-C5 treatment. *P < 0·05; **P < 0·01.

Fig. 4

Complement (C) activation is present at the end-plate in experimental autoimmune myasthenia gravis (EAMG). Muscle sections were stained for C3b (a–e) or C9/MAC (f–j) and double-stained with rhodamine–bungarotoxin (example in k,l). Representative fields for wild-type (a,f), Daf1^–/– (b,g), Cd59a^–/– (c,h) and Daf1^–/–CD59a^–/– (d,i) mice are shown. Plates (e,j) are representative sections for Daf1^–/–Cd59a^–/– mice treated with anti-C5 monoclonal antibody (mAb). Arrows indicate C3b/C9-positive bungarotoxin-positive end-plates. Magnification × 200; plates k,l × 1000.

Fig. 5

Muscle inflammation is exacerbated in Daf1^–/–CD59a^–/– mice. Muscle sections were stained with haematoxylin and eosin to identify inflammatory cell infiltrates. Representative fields for wild-type (a), Daf1^–/– (b), Cd59a^–/– (c) and Daf1^–/–CD59a^–/– (d) mice are shown. Plate (e) is a representative section from Daf1^–/–CD59a^–/– mice treated with anti-C5. Multiple sections were scored as described in Methods and the mean inflammatory score for each animal calculated. Plate (f) shows the summed data for each set in experiment 1; columns represent means of means and lines show standard deviations (n = 5 for each group). Plate (g) shows summed data for experiment 2 represented on the same scale as (f). For experiment 2, only four animals were analysed per group because of technical difficulties that rendered tissue from one animal unusable in the study. Here, too, columns represent means and lines show standard deviations. Magnification × 100. *P < 0·05; **P < 0·01.