Effect of anti-properdin IgG on C3 activation fragments and properdin deposition on apoptotic cells
Human endothelial cells from an umbilical vein (HUVEC) were used to study the possibility of anti-properdin to modulate the opsonization of apoptotic cells by the complement. The cell were characterized as late apoptotic cells after detection of phosphatidylserine on the cell membrane (binding to anexin V), impaired membrane permeability (permeability for propidium iodide) and DNA fragmentation (DAPI). The apoptotic cells were incubated with 1:10 diluted human sera mixed with IgG from LN patients with high levels of anti-properdin or anti-C3 autoantibodies. The dilution buffer contained 10 mM EGTA and 7 mM MgCl2 to allow activation only of the alternative pathway. Also, IgG from patients with anti-properdin positivity, but negative for anti-C1q, anti-C3b, anti-FB, anti-FH were selected for this experiment to minimize the confounding effects of other autoantibodies. EGTA-Mg allows activation of alternative complement pathway by inhibition of classical and lectin pathways. After incubation for 30 min/37°C the cells were labeled with mouse anti-C3c antibody (Quidel) or anti-properdin antibody (Quidel), diluted 1:50, followed by Alexa Fluor 555-conjugated anti-mouse IgG antibody (1:100) (Thermofisher). The cells were analyzed by FACS on an LSRII machine (BD Biosciences) and with FlowJo software.