Adrian Schreiber1, Friedrich C Luft, Ralph Kettritz. 1. HELIOS Klinikum-Berlin, Franz Volhard Clinic, and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany.
Abstract
BACKGROUND: Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response. METHODS: mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay. RESULTS: PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin. CONCLUSION: The degree of neutrophil mPR3 expression has definitive functional consequences.
BACKGROUND:Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response. METHODS:mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay. RESULTS:PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin. CONCLUSION: The degree of neutrophil mPR3 expression has definitive functional consequences.
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