title: Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies.
abstract: Antineutrophil cytoplasmic antibodies (ANCA) may be important in the pathophysiology of necrotizing vasculitis. ANCA activate human neutrophils primed with tumor necrosis factor-alpha (TNF-alpha) in vitro. TNF-alpha priming results in translocation of ANCA antigens to the cell surface, where they are recognized by the antibodies. The signaling mechanisms involved in TNF-alpha priming and subsequent ANCA-induced activation were investigated. TNF-alpha-primed neutrophils were stimulated with monoclonal antibodies (MAb) to human myeloperoxidase (MPO) and proteinase 3 (PR3), and with preparations of human ANCA (three patients with PR3-ANCA and two patients with MPO-ANCA). Respiratory burst was measured with superoxide dismutase-inhibitable ferricytochrome C reduction and using dihydro-rhodamine-1,2,3. Phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) and the extracellular signal-regulated kinase (ERK) were assessed by immunoblotting. ANCA-antigen translocation was studied by flow cytometry. The tyrosine phosphorylation inhibitor genistein, but not calphostin or staurosporin, resulted in a significant dose-dependent superoxide generation inhibition (11.6 +/- 1.7 nmol to 2.1 +/- 0.5 for PR3-ANCA, and 16.0 +/- 2.8 to 3.3 +/- 1.3 for MPO-ANCA). The p38-MAPK inhibitor (SB202190) and the ERK inhibitor (PD98059) diminished PR3-ANCA-mediated superoxide production dose dependently (11.6 +/- 1.7 nmol O(2)(-) to 1.9 +/- 0.6 with 50 microM SB202190 and 4.0 +/- 0.6 with 50 microM PD098059, respectively). For MPO-ANCA, the results were similar (16.0 +/- 2.8 nmol to 0.9 +/- 1.0 nmol with SB202190 and 6.4 +/- 2.4 nmol with PD98059, respectively). Western blot showed phosphorylation of both p38-MAPK and ERK during TNF-alpha priming. The p38-MAPK inhibitor and the ERK inhibitor showed the strongest effect on respiratory burst when added before TNF-alpha priming, further supporting an important role for both signaling pathways in the priming process. Flow cytometry showed that p38-MAPK inhibition decreased the translocation of PR3 (by 93 +/- 2%) and of MPO (by 64 +/- 2%). In contrast, no such effect was seen when ERK was inhibited. Thus, p38-MAPK and ERK are important for the TNF-alpha-mediated priming of neutrophils enabling subsequent ANCA-induced respiratory burst. However, both pathways show differential effects, whereby p38-MAPK controls the translocation of ANCA antigens to the cell surface.
authors.affiliation: Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Kettritz
authors.affiliation: Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Schreiber
authors.affiliation: Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
authors.forename: Friedrich C
authors.initials: FC
authors.lastname: Luft
authors.affiliation: Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Haller
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053146:Myeloblastin; D009281:Naphthalenes / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology*; D009195:Peroxidase / Q000276:immunology; D010766:Phosphorylation; D016897:Respiratory Burst / Q000187:drug effects; D012697:Serine Endopeptidases / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D009281:Naphthalenes; D014409:Tumor Necrosis Factor-alpha; C061465:calphostin complex; D013481:Superoxides; D014443:Tyrosine; D019833:Genistein; D009195:Peroxidase; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D012697:Serine Endopeptidases; D053146:Myeloblastin; D019311:Staurosporine