FcgammaRII-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 FcgammaRII and the glycan phosphoinositol-linked FcgammaRIIIB. Use of monoclonal antibodies that distinguished the two Fc receptors demonstrated that FcgammaRII ligation was 100-fold more potent at signaling L-plastin phosphorylation than occupancy of FcgammaRIIIB. Depletion of intracellular calcium did not affect FcgammaRII-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')2, 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 FcgammaRII-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.
Jones
S L
SL
Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Brown
E J
EJ
D000185:Actinin; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology; D018821:CD18 Antigens / Q000276:immunology; D002118:Calcium / Q000097:blood; D002448:Cell Adhesion; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007700:Kinetics; D018370:Leukocyte-Adhesion Deficiency Syndrome / Q000097:blood / Q000276:immunology; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D010587:Phagocytosis; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D017452:Receptors, IgG / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016596:Vinculin
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D017452:Receptors, IgG; C026742:plastin; D000185:Actinin; D016596:Vinculin; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium