Anti-PLA2R1 Antibodies Containing Sera Induce In Vitro Cytotoxicity Mediated by Complement Activation

Figure 2

Anti-PLA2R1-mediated cytotoxicity depends on complement in immunofluorescence cytotoxicity assay. (a) Anti-PLA2R1-mediated cytotoxicity depending on complement for a PLA2R1-positive serum and a healthy donor serum. HEK T+: HEK293T-REx cells transfected with PLA2R1 and induced with tetracycline to overexpress PLA2R1. HEK T-: HEK293T-REx cells transfected with PLA2R1 but not induced with tetracycline. C+: addition of rabbit complement. C-: no addition of rabbit complement. Dead cells appear in red; living cells appear in green. (b) Anti-PLA2R1 complement-mediated cytotoxicity in a cohort of 48 patients and 20 healthy donors in different conditions. Note a low level of cytotoxicity in noninduced HEK that tended to be significant () caused by a minimal expression of PLA2R1 even in noninduced cells, as determined by western blot (c). All samples were tested using the same batch of HEK293 cells. A minimum of 50 cells per well was necessary for reading. (c) Expression of PLA2R1 in noninduced and induced HEK293 cells with tetracycline. Note a minimal expression of PLA2R1 in noninduced cells. (d) Analysis of complement activation pathways involved in anti-PLA2R1-mediated cytotoxicity for 3 PLA2R1-positive patients using two inhibitors of complement pathways. Patients 1 and 2 were positive for both IgG3 and IgG4 anti-PLA2R1 antibodies, while patient 3 was only positive for IgG4 anti-PLA2R1. An excess of GVB-EDTA (inhibitor of the three pathways of the complement) or Mg-EGTA (inhibitor of the classical and lectin pathways) was added in serum + complement and the complement-mediated cytotoxicity was measured. All samples were tested using the same batch of HEK293 cells. A minimum of 50 cells per well was necessary for reading. (e) Analysis of complement activation pathways involved in anti-PLA2R1-mediated cytotoxicity for 3 PLA2R1-positive patients. Note that GVB-EDTA strongly inhibits anti-PLA2R1-mediated cytotoxicity in all 3 patients, while Mg-EGTA, which is known to inhibit only the classical and lectin pathways, inhibits only partially the anti-PLA2R1-mediated cytotoxicity, suggesting a potential activation of the alternative pathway in some serum samples. A minimum of 50 cells per well was necessary for reading. HEK293 T-REx cells transfected with PLA2R1 and induced with tetracycline to express PLA2R1 were used. Negative controls (noninduced HEK T-REx cells) are not shown. All cytotoxicity assays were performed using the same batch of HEK293 T-Rex cells. A minimum of 50 cells per well was necessary for reading.

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