Fig. 5
Complement activation by IgM anti-GM2 bound to SCs in culture. (A) Representative microscopic images of complement activation on SCs opsonized with IgM anti-GM2 positive MMN serum or not, and incubated with fresh serum, heat-inactivated serum (serum HI), or serum pre-incubated with a monoclonal antibody that blocks C5 activation or an isotypic control antibody, as complement source. 20x magnification, scale bar: 50 μm. (B) Quantification of microscopy data using 3 different MMN sera for opsonization.C3 fixation to the cells (quantified as MGV) is expressed as FC (FCC3) setting the fixation observed with cells not opsonized with MMN serum (striped bar) as 1. Opsonization of the cells with MMN serum results in a significant increase in C3 fixation, which is abrogated when heat inactivated serum is used as complement source, and which is also reduced wen the complement source is pre-incubated with anti-C5 antibody, and not with an isotypic control antibody. (C) Quantification of C5a, depicted as FCC5a, measured in the supernatant of SC cultures. C5a generation in culture medium is increased upon opsonization of the cells with IgM anti-GM2 positive MMN patient serum and incubation with fresh serum. This C5a generation is inhibited by an anti-C5 antibody added to the complement source but not by a control antibody. Mean + SD, Kruskal-Wallis, post-hoc Dunn’s multiple comparisons test, ** p < 0.01; **** p < 0.0001; FC: fold change; HI: heat inactivated; MMN: multifocal motor neuropathy; ns: non-significant