Effect of allotype on activation of neutrophils by FcgammaRIIIB cross-linking.

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