Fc gamma RII-mediated adhesion and phagocytosis induce L-plastin phosphorylation in human neutrophils

L-Plastin is a calcium-regulated actin bundling protein expressed in leukocytes and some transformed cells, which is phosphorylated on serine in response to several different leukocyte-activating stimuli. Adhesion to immune complexes induced L-plastin phosphorylation in neutrophils, as did phagocytosis of IgG-opsonized particles, but insoluble immune complexes in suspension were very inefficient activators of L-plastin phosphorylation. Neutrophils express two IgG Fc receptors, the transmembrane Fc gamma RII and the glycan phosphoinositol-linked Fc gamma RIIIB. Use of monoclonal antibodies that distinguished the two Fc receptors demonstrated that Fc gamma RII ligation was 100-fold more potent at signaling L-plastin phosphorylation than occupancy of Fc gamma RIIIB, Depletion of intracellular calcium did not affect Fc gamma RII-activated L-plastin phosphorylation, demonstrating that any potential regulation of plastin function by calcium did not affect its phosphorylation, Adhesion to immune complexes caused L-plastin to localize to podosomes, since it colocalized with actin to discrete, punctate Triton X-100-insoluble sites on the adherent neutrophil surface in a pattern indistinguishable from vinculin and alpha-actinin, Nonetheless, localization to podosomes was not required for L-plastin phosphorylation, since both neutrophils from a patient with leukocyte adhesion deficiency (CD18 deficiency) and neutrophils treated with anti-CD18 F(ab')a, which do not form podosomes upon adhesion to immune complexes, phosphorylated L-plastin normally, Indeed, L-plastin was normally phosphorylated in response to adhesion to immune complexes even when the actin cytoskeleton was disrupted with cytochalasin D. We conclude that efficient Fc gamma RII-mediated phosphorylation of L-plastin requires cell adhesion but does not require IgG-induced rearrangements of the actin cytoskeleton. These data suggest a model in which plastin phosphorylation and localization to the actin cytoskeleton can act as two distinct mechanisms regulating L-plastin functions in neutrophils adherent to immune complexes.