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.