title: Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation.
abstract: The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.
authors.affiliation: Medical Faculty of the Charité, Experimental and Clinical Research Center, Berlin, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Jerke
authors.forename: Susanne
authors.initials: S
authors.lastname: Rolle
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.forename: Behnaz
authors.initials: B
authors.lastname: Bayat
authors.forename: Sentot
authors.initials: S
authors.lastname: Santoso
authors.forename: Anje
authors.initials: A
authors.lastname: Sporbert
authors.forename: Friedrich
authors.initials: F
authors.lastname: Luft
authors.forename: Ralph
authors.initials: R
authors.lastname: Kettritz
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000276:immunology / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007519:Isoantigens / Q000276:immunology / Q000378:metabolism*; D016177:Macrophage-1 Antigen / Q000276:immunology / Q000378:metabolism*; D053146:Myeloblastin / Q000276:immunology / Q000378:metabolism*; D018375:Neutrophil Activation / Q000502:physiology; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D016897:Respiratory Burst / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology*; D013481:Superoxides / Q000276:immunology / Q000378:metabolism; D014657:Vasculitis / Q000276:immunology / Q000378:metabolism
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; C407242:CD177 protein, human; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D007074:Immunoglobulin G; D007519:Isoantigens; D016177:Macrophage-1 Antigen; D011956:Receptors, Cell Surface; D013481:Superoxides; D053146:Myeloblastin