Effect of Allotype on Activation of Neutrophils by FcγRIIIB Cross-Linking

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcγRII, and FcγRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcγR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+]i) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD–phallacidin using flow cytometry. Cross-linking of FcγRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+]i, superoxide generation, and the polymerization of actin. [Ca2+]i responses from cross-linking of FcγRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+]i release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcγRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+]i response and actin polymerization induced by FcγRIIIB cross-linking. Cross-linking of FcγRIIIB by 3G8 produced minimal [Ca2+]i release and polymerization of actin irrespective of donor allotype.