Figure 2

Cross- linking of IgGs bound to human neutrophils leads to FcγR-dependent tyrosine phosphorylation and calcium mobilisation. A: Isolated neutrophils were incubated with 10% autologous PPP with or without FcγRs blocking monoclonal antibodies as described in Methods and Figure 1. Cross-linking of surface-bound IgGs was achieved by the addition of anti-human IgG antibodies. The reaction was stopped by transferring aliquots of the neutrophil suspensions directly in the same volume of boiling 2X concentrated modified Laemmli’s buffer [33]. Whole cell lysates (2.5x10⁵ cells in each lane) were analysed by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody 4G10 or anti-actin antibody (loading control). B: Isolated neutrophils were preincubated with Fura2-AM, followed by an incubation with or without FcγRs blocking antibodies and with 10% autologous PPP. Anti-human IgG antibodies were added and fluorescence was monitored as described in Methods. C: Cross-linking of surface-bound IgGs was achieved by the addition of either complete anti-human IgG antibodies or F(ab’)₂ fragment of anti-human IgG antibodies. D: Neutrophils were incubated with or without PPP before cross-linking of surface bound IgGs. Results are representative of, at least, three independent experiments.