Figure 6

Rheumatoid factors induce FcγRs- dependent adhesion of neutrophils to HUVECs. A: An aliquot of the preparation of rheumatoid factors (100 ng and 10 ng) was analyzed by SDS-PAGE followed by immunoblotting with horseradish peroxidase-labelled anti-human IgG or anti-human IgM antibodies. The same amounts of commercial human IgG and IgM were analyzed on the same gel. The IgM band was used for quantification (arrow). B: Neutrophils were incubated with calcein and with or without PPP, as described in Methods. Neutrophils were then plated on a confluent monolayer of endothelial cells and RFs (500 μg/ml) were added for 2 hours. Non-adherent neutrophils were discarded and the amount of adherent cells was quantified as the amount of fluorescence bound to endothelial cells. Results are expressed as mean +/- SEM of three independent experiments. ** = P < 0.005, *** = P < 0.0005 (one-sample t test). C: Heat aggregated IgGs (HA-IgGs) were prepared as previously described [41]. Neutrophils were incubated with calcein, plated on endothelial cells and 100 μg/ml HA-IgG (HA) were added for 2 hours. Results are expressed as mean +/- SEM of three independent experiments, each carried out in triplicate. ** = P < 0.005, *** = P < 0.0005 (Mann–Whitney test).