title: Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab')2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG.
abstract: Anti-neutrophil cytoplasm autoantibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. Intact ANCA IgG activate superoxide generation in cytokine-primed neutrophils after binding their antigens and co-engaging Fcgamma receptors (FcgammaR). The contribution of antigen binding via ANCA F(ab')(2) fragments to signaling has been unclear. This study shows that both ANCA IgG and F(ab')(2) fragments of ANCA IgG induce significant GTPase activity, which could be blocked with pertussis toxin and anti-G(i) protein antibodies. Pertussis toxin inhibited ANCA IgG-induced superoxide generation but was without effect on superoxide production after conventional FcgammaR ligation. ANCA F(ab')(2) fragments did not induce superoxide generation. ANCA IgG activated PI 3-kinase-generating PIP(3), activated protein kinase B (PKB), and p21(ras); activation of each mediator was inhibited with pertussis toxin, but PI3K and PKB were not activated by ANCA IgG F(ab')(2) fragments. Intact ANCA IgG induced tyrosine phosphorylation, whereas F(ab')(2) fragments did not, and ANCA IgG-mediated superoxide generation was inhibited with genistein. Both genistein and pertussis toxin together completely abrogated the ANCA-induced oxidative burst. Genistein also inhibited ANCA IgG-induced PIP(3) generation and p21(ras) activation. These data implicate a novel ANCA IgG stimulated signaling pathway that involves both F(ab')(2)-mediated antigen binding and Fc-mediated FcgammaR ligation in cooperative interactions between G(i) proteins and tyrosine kinases that facilitates activation of downstream mediators.
authors.affiliation: Renal Immunobiology, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, UK.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Williams
authors.forename: Anne
authors.initials: A
authors.lastname: Ben-Smith
authors.forename: Peter
authors.initials: P
authors.lastname: Hewins
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Dove
authors.forename: Philip
authors.initials: P
authors.lastname: Hughes
authors.forename: Robert
authors.initials: R
authors.lastname: McEwan
authors.forename: Michael J O
authors.initials: MJ
authors.lastname: Wakelam
authors.forename: Caroline O S
authors.initials: CO
authors.lastname: Savage
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000276:immunology; D013481:Superoxides / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000378:metabolism
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D004791:Enzyme Inhibitors; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; C118303:phosphoinositide-3,4,5-triphosphate; D020703:ras GTPase-Activating Proteins; D013481:Superoxides; D014443:Tyrosine; D019833:Genistein; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019206:GTP-Binding Protein alpha Subunits, Gi-Go